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134 Cards in this Set

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What makes red blood cells red?

Red blood cells are very abundant, carries oxygen to tissue and CO2 away from tissues and the membrane is colourless.



The red comes from hemoglobin which is a protein molecule that carries oxygen to tissues and very abundant . Hemoblogin contains a heme, an iron binding component of hemoglobin, which gives it its red colour.

What is hemoglobin?

Oxygen transport protein of erythrocytes, is a tetramer composed of 2 alpha-chains and 2 beta-chains 
 
Heme = iron binding component of hemoglobin
(Heme is a prosthetic group)
 
Structure was Found in 1950s 

Two structures 
 
-Heme:
• Iron bin...

Oxygen transport protein of erythrocytes, is a tetramer composed of 2 alpha-chains and 2 beta-chains



Heme = iron binding component of hemoglobin


(Heme is a prosthetic group)



Structure was Found in 1950s



Two structures



-Heme:
• Iron binding component in hemoglobin and • needed to make hemoglobin
needed to make it which gives its red colour
• Is a prosthetic group consisting if Fe2+



-Globin
• The protein



4 subunits, tetramer
-2 identical alpha-chains
-Two identical beta-chains



The spirals are alpha helices
• This is an alpha helical contained protein



Some aa have very little affect when you change while some causes diseases

What is sickle cell anemia?

A single amino acid change (Glu6Val) in beta-chain of hemoglobin causes sickle cell anemia


• A single change in aa causes this change, the Glu protein in the sixth position to a Val which resulted in a human diseases



Hb S forms long polymers that change the shape of red blood cells



Anemia means lower red cell count



What is a protein?

Proteins are polymers of amino acids linked in series by peptide bonds.


• Linear sequence of amino acids



Proteins fold into specific 3D structures


• Not linear chain but folds into 3D structures and different structures/fold have different functions or not working properly when misfolded


• When proteins are misfolded they turn back into aa's and are chopped up and recycled again



Agents of biological function, often enzymes


• Enzymes are catalyzes and speed up reactions, carbonic anhydrase catalyzes into H2O a million times per second per molecule



Encoded by genes



Mutations in genes lead to defective proteins and inherited diseases

What is H2O and its properties?

Water = H2O



• Water is polarized where H is EN+ while O is EN-, it is dipole



• Oxygen loves to interact with itself using hydrogen bonds



• Water is attracted to each other and hydrogen bonds are constantly being broken and created, especially depending temperature



• The oxygen can form weak hydrogen bonds where it can also accept hydrogen bonds because it is EN-



• Without water, proteins won't be able to fold up properly.



• Proteins are designed to fold in aqueous solutions



• Proteins will NOT fold up in oil



• Most important molecule in biology and essential for life as we know it



• Water has a higher boiling point, melting point, heat of vaporization and surface tension compared to other hydrides



• The potential to form 4 H bonds per water molecule is the source of the strong intermolecular attractions that endow this substance with its anomalously high boiling point, melting point, heat of vaporization and surface tension



• Each H2O molecule has 4 nearest neighbours to which it is hydrogen bonded: Each H atom donates a H bond to the O of a neighbor and the O atom serves as a H-bond acceptor from H atoms bound to 2 different molecules



• H-bonded water molecule serving as an acceptor is a better H-bond donor than an unbounded molecule and a H2O molecule serving as a H-bond donor becomes a better H-bond acceptor



• The directional preference of H bonds leads to an open lattice structure



• The presence of strain creates a kinetic situation in which H2O molecules can switch H-bond allegiances; fluidity ensues

What are hydrophobic interactions?

Non-polar groups are driven together not so much because they have a high affinity for each other but because water hydrogen bonds strong to itself



Hydrophobic interactions result from the strong tendency of water to exclude non-polar groups or molecules



They arise because water molecule prefer the stronger interactions that they share with one another compared to their interaction with non-polar molecules



Non-polar solutes do not readily H bond to H2O and as a result, such compounds tend to be only sparingly soluble in water



The process of dissolving such substances is accompanied by significant reorganization of the water surrounding the solute so that the response of the solvent water to such solutes can be equated to "structure making"



Consequently, the H-bonded water network rearranges towards formation of a local cage-like (clathrate) structure surrounding each solute molecule



This fixed orientation of water molecules around a hydrophobic "solute" molecule results in a hydration shell



Clathrate formation is accompanied by significant ordering of structure or negative entropy



Hydrophobic effect happens inside the protein

What is ionic interactions?

Opposite charges attract each other



The result of attractive forces between oppositely charged structures, such as negative carboxyl groups and positive amino groups



Strength of electrostatic interactions is highly dependent on the nature of interacting species and the distance, r, between them



• May involve ions (species possessing discrete charges), Permanent dipoles (having a permanent separation of positive and negative charges) or induced dipoles (having temporary separation of positive and negative charge induced by the environment

What are hydrogen bonds?

Specific



Has hydrogen, hydrogen acceptor who is usually negative and shared and very linear and very specific



Water can break hydrogen bonds as they are always in constant competition with each other



Hydrogen bonds form between a hydrogen atom covalently bonded to an electronegative atom (such as O or N) and a second EN negative atom that serves as the hydrogen bond acceptor



Stabilizing, attractive interactions between a proton and an electron - donating species

What are Van der Waals interactions?

• Contact



• Protein are tightly packed like a crystal, so packed so tightly and the interaction between the nucleus and electrons of another, very weak and insignificant



• Van der Waals forces are the result of induced electrical interactions between closely approaching atoms or molecules as their negatively charged electron clouds fluctuate instantaneously in time



• They operate only over a very limited interatomic distance (0.3 - 0.6 nm) and are an effective bonding interaction at physiological temperatures only when a number of atoms in a molecule can interact with several atoms in a neighbouring molecule



• Tend to be weak and individually contribute 0.4 - 4.0 kJ/mol of stabilization energy

What are olgiosaccharides?

Polymers of sugars linked in linear and branched series of glycosidic bonds resulting in complex structures

What are the four nucleotide bases of DNA?

2 Purines:


-Adenine


-Guanine



2 Pyrimidines:


-Thymine


-Cytosine

What is another name for alpha-carbons and what is special about them?

Central carbon



Alpha-carbon are asymmetric or chiral and the 2 possible configurations for the alpha constitute non-superimposable mirror-image isomers, or enantiomers

How are peptide bonds formed and what are they?

• Amino acids can join via peptide bonds 
 
• The crucial feature of amino acids that allow them to polymerize to form peptides and proteins in the existence of their 2 identifying chemical groups: the amino (-NH3+) and carboxyl (-COO-) groups...

Amino acids can join via peptide bonds



The crucial feature of amino acids that allow them to polymerize to form peptides and proteins in the existence of their 2 identifying chemical groups: the amino (-NH3+) and carboxyl (-COO-) groups



They can react in head-to-tail fashion, eliminating a water molecule and forming a covalent amide linkage, which, in the case of peptide and proteins, is typically referred to as a peptide bond



Equilibrium of reaction favours peptide bond hydrolysis



Is planar and trans between carbonyl O and amide H



Dihedral angel w about the peptide C-N bond is fixed at 180 degrees



Although it is 2 planes, when joined together, it can be rotated (turn about alpha carbon)

What is zwitterion?

A dipolar ion



Had negative and positive change creating a neutral molecule (no net charge)

How do you know if a beta carbon is NOT beta branched?

It has 2 or more hydrogens bonded to it

What is Glycine structure, short form and properties?

Glycine, Gly, G


 


• Only has a hydrogen atom as a side chain 


 


• Aliphatic side chain


 


• Does not have an L or D forms 


 


• Very and maybe most flexible aa and the smallest 


 


• Allow...

Glycine, Gly, G



• Only has a hydrogen atom as a side chain



• Aliphatic side chain



• Does not have an L or D forms



• Very and maybe most flexible aa and the smallest



• Allows close packing and van de Waal forces
Hydrophobic



• Simplest amino acid, has only a single hydrogen for an R group and this hydrogen is not a good hydrogen bond former,



• Glycine's solubility properties are influenced mainly by its polar amino and carboxyl groups and thus glycine is best considered a member of the polar, uncharged group



• Except for glycine, all the amino acids isolated from proteins have 4 different groups attached to the alpha-carbon atom



• Glycine is sterically the most adaptable of the amino acids and it accommodates conveniently to other steric constraints in the beta-turn

What is Alanine structure, short form and properties?

Alanine, Ala, A


 


• Has a methly group for R-chain


 


• Aliphatic side chain


 


• Most generic


 


• Non-polar


 


• Hydrophobic

Alanine, Ala, A



Has a methly group for R-chain



Aliphatic side chain



Most generic



Non-polar



Hydrophobic

What is Valine structure, short form and properties?

Valine, Val, V


 


• Beta branched


 


• Large aliphatic chain


 


• Non-polar


 


• Hydrophobic due to aliphatic chain


 


• Terrible alpha-helix former due to beta branch

Valine, Val, V



• Beta branched



• Large aliphatic chain



• Non-polar



• Hydrophobic due to aliphatic chain



• Terrible alpha-helix former due to beta branch

What is Leucine structure, short form and properties?

Leucine, Leu, L


 


• Aliphatic side chain


 


• Most common amino acid in proteins


 


• Great alpha-helix former


 


• Hydrophobic


 


• Non-polar

Leucine, Leu, L



Aliphatic side chain



Most common amino acid in proteins



Great alpha-helix former



Hydrophobic



Non-polar

What is Isoleucine structure, short form and properties?

Isoleucine, Ile, I


 


• Aliphatic side chain


 


• Non-polar


 


• Hydrophobic

Isoleucine, Ile, I



• Aliphatic side chain



• Non-polar



• Hydrophobic

What is Serine structure, short form and properties?

Serine, Ser, S


 


• Has a hydroxyl group: 


-Form hydrogen bonds with water where the Oxygen can be a hydrogen acceptor which makes it polar 

-Can also interact with other polar groups 


 


• Aliphatic hydroxyl side chai...

Serine, Ser, S



• Has a hydroxyl group:


-Form hydrogen bonds with water where the Oxygen can be a hydrogen acceptor which makes it polar
-Can also interact with other polar groups



• Aliphatic hydroxyl side chain



• Good hydrogen bond-forming moeities



• Hydrophilic



• Polar



• EN negative

What is Threonine structure, short form and properties?

Threonine, Thr, T


 


• Beta branched


 


• Aliphatic hydroxyl side chain


 


• Polar


 


• EN negative


 


• Hydroxyl group AND methyl group


 


• Good hydrogen bond-forming moieties


 

...

Threonine, Thr, T



• Beta branched



• Aliphatic hydroxyl side chain



• Polar



• EN negative



• Hydroxyl group AND methyl group



• Good hydrogen bond-forming moieties



• Hydrophilic

What is Phenyalanine structure, short form and properties?

Phenyalanine, Phy, F


 


• Aromatic side chain


 


• Benzyl group R-side chain


 


• Hydrophobic 


 


• Absorb ultraviolet light above 250nm

Phenyalanine, Phe, F



• Aromatic side chain



• Benzyl group R-side chain



• Hydrophobic



• Absorb ultraviolet light above 250nm

What is Tyrosine structure, short form and properties?

Tyrosine, Tyr, R


 


• Aromatic side chain


 


• Amphipathic


 


• Hydrophobic with polar properties


 


• Good hydrogen-bond forming moieties


 


• Also has non-polar characteristics due to its aromati...

Tyrosine, Tyr, Y



• Aromatic side chain



• Amphipathic



• Hydrophobic with polar properties



• Good hydrogen-bond forming moieties



• Also has non-polar characteristics due to its aromatic ring and could be arguably be placed in the non-polar group


(Has pKa of 10.1, its phenolic hydroxyl is a charged, polar entity at high pH)



• Absorb ultraviolet light above 250nm

What is Tryptophan structure, short form and properties?

• Aromatic side chain


 


• Has indole ring R-side chain which gives it absorption of 290nm light


 


• Nitrogen on indole ring give it hydrogen donor potential


 


• Considered a borderline member of aromatic side cha...

Tryptophan, Trp, W



• Aromatic side chain



• Has indole ring R-side chain which gives it absorption of 290nm light



• Nitrogen on indole ring give it hydrogen donor potential



• Considered a borderline member of aromatic side chain group because it can interact favourable with water via the N-H moiety of indole ring

What is Beer-Lambert Law?

Concentration of a give target protein solution is directly related to its UV absorption reading



A=ecl



Where A is absorbance, e is moral extinction coefficient, c is concentration of target protein and l is length of optical path

What is Cysteine structure, short form and properties?

Cysteine, Cys, C


 


• Contains sulfur


 


• EN negative


 


• Can deprotonate at pH values greater than 7


 


• Hydrophilic


 


• Can create disulfide bridges with other cysteines 

Cysteine, Cys, C



Contains sulfur



EN negative



Can deprotonate at pH values greater than 7



Hydrophilic



Can also be considered hydrophobic because of its sulfide and found buried inside the protein



Can create disulfide bridges with other cysteines

What is Methionine structure, short form and properties?

Methionine, Met M


 


• Contains sulfur


 


• EN negative


 


• Often the first protein to be cut off as it is the initiator protein


 


• Amphipathic


(least polar of the amphipathic amino acids but its thio...

Methionine, Met M



Contains sulfur



EN negative



Often the first protein to be cut off as it is the initiator protein



Amphipathic


(least polar of the amphipathic amino acids but its thioether sulfur can be an effective metal ligand in proteins)



Hydrophobic

What is a disulfide bridge and what is its properties?

• It is a link between two cysteine amino acids put together in a protein


 


• There is an enzyme that breaks the disulfide bridge over and over again till protein gets it right


 


• Disulfide bridges exist outside and insid...

• It is a link between two cysteine amino acids put together in a protein



• There is an enzyme that breaks the disulfide bridge over and over again till protein gets it right



• Disulfide bridges exist outside and inside of cells



• Increase stability of proteins



• This is a reduced form, if you oxidize it, you create a disulfide bridge and you can reduce it to break the bridge



• Formed during protein folding process



• Two cysteine from other sides of protein can bind together and help anchor the protein

What is Asparate structure, short form and properties?

Aspartate, Asp, D


 


• Polar


 


• Acidic


 


• Hydrophilic

Aspartate, Asp, D



• Polar



• Acidic



• Hydrophilic

What is Glutamate structure, short form and properties?

Glutamate, Glu, E


 


• Polar


 


• Acidic

Glutamate, Glu, E



• Polar



• Acidic

What is Asparagine structure, short form and properties?

Asparagine, Asn, N


 


• Good hydrogen-bonding forming moieties 


 


• Hydrophilic 


 


• To test what a D is doing in a protein, you can change it to N to see if it does anything or if it kills the protein


 

...

Asparagine, Asn, N



• Good hydrogen-bonding forming moieties



• Hydrophilic



• To test what a D is doing in a protein, you can change it to N to see if it does anything or if it kills the protein



• Polar



• Amide R-side chain



• Acidic

What is Glutamine structure, short form and properties?

Glutamine, Gln, Q


 


• Hydrophilic


 


• Acidic

Glutamine, Gln, Q



Hydrophilic



Acidic

What is Lysine structure, short form and properties?

Lysine, Lys, K


 


Has a 4 methyl group in a row making it hydrophobic 


• Head poking out interacting with water while the chain is hiding inside the protein, called snorkeling 


• Head is different from rest of protein 


...

Lysine, Lys, K



Has a 4 methyl group in a row making it hydrophobic


• Head poking out interacting with water while the chain is hiding inside the protein, called snorkeling


• Head is different from rest of protein



Basic



Lysine contains a protonated alkyl amino group



Side chains are protonated under physiological conditions and participate in electrostatic interactions in proteins



Amphipathic



Can be considered amphipathic because its R group consists of an aliphatic side chain which can interact with hydrophobic amino acids in protein and normally charged at neutral pH



Polar

What is Arginine structure, short form and properties?

Arginine, Arg, R


 


Very positively charge and never lose it under natural circumstances 


 


Very important for binding substances 


 


Basic 


 


Arginine contains aguanidiniumgroup 


 


Side chains are pr...

Arginine, Arg, R



Very positively charge and never lose it under natural circumstances



Very important for binding substances



Basic



Arginine contains aguanidiniumgroup



Side chains are protonated under physiological conditions and participate in electrostatic interactions in proteins



Hydrophilic



Has resonance structure due to double bond


What is Histidine structure, short form and properties?

Histidine, His, H


 


Has an imidazole ring where resonance structure where the positive charge can change. It can lose it proton easily where it can take or lose easily 


 


Basic  


 


Side chains fully protonated at pH7...

Histidine, His, H



Has an imidazole ring where resonance structure where the positive charge can change. It can lose it proton easily where it can take or lose easily



Basic



Side chains fully protonated at pH7 but histidine with a side chainpKaof 6 which means it is only 10% protonated at pH 7



With a pKa near neutrality, histidine side chains plays important roles as proton donors and acceptors in many enzyme reaction



Hydrophilic

What is Proline structure, short form and properties?

Proline, Pro, P


 


Is an imino acid, not really an amino acid but is because it is cyclic


 


Side chain is cyclic and forms a ring via a covalent bond with the backbone nitrogen atom.


 


Cyclic ring makes it a very rigid st...

Proline, Pro, P



Is an imino acid, not really an amino acid but is because it is cyclic



Side chain is cyclic and forms a ring via a covalent bond with the backbone nitrogen atom.



Cyclic ring makes it a very rigid structure and makes the kink in chains



If you put in alpha helix, it would break it and bend it



For the protein to fold, it is often found that proline is found in the bends and folds



Non-polar



Hydrophobic



Proline has a cyclic structure and a fixed phi angel, so, to some extent, it forces the formation of a beta-turn

What are the hydrophobic amino acids?

Ala, Cys, Ile, Leu, Met, Phe, Val

What are the hydrophilic amino acids?

Basic: Arg, Lys



Acidic: Asp, Glu



Polar: Asn, Gln, His

What is the most common amino acid?

Leu

What is pH?

Soren Sorensen devised the pH scale by defining pH as the negative logarithm of the hydrogen ion concentration:



pH = -log[H+]


pKw = pH + pOH = 14



Low pH values represent the highest H+ concentration and lowest OH- concentrations



Point of neutrality is at pH 7 and this is called neutral pH



Substances that are almost completely dissociated to form ions in solutions are called strong electrolytes



Electrolyte describes substances capable of generating ions in solution and thereby causing an increase in the electrical conductivity of the solution



Acids are proton donors and bases are proton acceptors



Substances with only slight tendency to dissociate to form ions in solution are called weak electrolytes



Ka is also termed an ionization constant because it states the extent to which a substance forms ions in water



Relatively low values of Ka for acetic acid reveals that un-ionized form, CH3COOH, predominates over H+ and CH3COO- in aqueous solutions of acetic acid

What is the Henderson-Hasselbalch equation?

Describe the Dissociation of a Weak Acid in the Presence of its conjugate base



pH = pKa + log([A-]/[HA])



The pH of a solution can be calculated provided Ka and the concentration of the weak acid HA and its conjugate based A- are known

What is the pKa's of R, K, C, H, E, D?

R = 12.5


 


K = 10.5


 


C = 8.3


 


H = 6.0


 


E = 4.3


 


D = 3.9

R = 12.5



K = 10.5



C = 8.3



H = 6.0



E = 4.3



D = 3.9

What is Trypsin?

Digestive enzyme trypsin is the most commonly used reagent for specific proteolysis 


 


Will only hydrolyze peptide bonds in which the carbonyl function is contributed by an arginine or a lysine residue 


 


Trypsin cleaves on the...

Digestive enzyme trypsin is the most commonly used reagent for specific proteolysis



Will only hydrolyze peptide bonds in which the carbonyl function is contributed by an arginine or a lysine residue



Trypsin cleaves on the C-side of Arg or Lys, generating a set of peptide fragments having Arg or Lys at their C-termini



Number of small peptides resulting from trypsin action is equal to the total number of Arg and Lys residues n the protein plus one - the protein's C-terminal peptide fragment

What is protein primary structure?

Amino acid sequence

What is protein secondary stucture?

Periodic, repetitive units (alpha-helix, beta-sheet, reverse turn)



In the alpha-helix, the N-H group of residue (i) donates a H-bond back to C=O group of residue (i - 4)



Need to pack side chains and minimize side-chain steric clashes



Through hydrogen-bonding interactions between adjacent amino acid residues



Is the 3D arrangement

What is protein tertiary structure?

Assembly of secondary + unstructured segments into functional protein



Defines the final folded functional structure of the protein



Represents the assembly of alpha helices, beta sheets, beta turns, intervening and disulfide bonds



Segments without periodicity are described as 'random' or 'unstructured'



Structure defined by sequence



Proteins are dynamic and exist in different conformational states

What is protein quaternary structure?

Assembly of protein sub-units



Described proteins where function depends upon the assembly of two or more subunits



Subunits interact through the same forces as occur within individual subunits



Subunits can be identical, or may all be different



Key role played by van der Waals packing and hydrophobic effect

What is an alpha-helix?

Intramolecular H-bonds stabilize the structure:


 


N-H group of residues i donates hydrogen back to C = O of residue i - 4, instead of H-bonding with water


 


Secondary structure


 


Each peptide bond posses a dipole moment ...

Intramolecular H-bonds stabilize the structure:



N-H group of residues i donates hydrogen back to C = O of residue i - 4, instead of H-bonding with water



Secondary structure



Each peptide bond posses a dipole moment that arises from the polarities of the N-H and C=O groups and because these groups are all aligned along the helix axis, the helix itself has a substantial dipole moment with a partial positive charge at the N-terminus and a partial negative charge at the C-terminus



In a typical alpha helix, there are ( n - 4 ) hydrogen bonds



3.6 residues per 360 degrees turn (3 aa during single turn)



100 degrees between adjacent residues.



Side chains exposed around the periphery of the structure



Most helices are amphipathic

What is a pitch in an alpha-helix?

Translation distance of the helix



If ignoring side chains, the helix is about 6 A in diameter



Side chains, extending outward from the core structure of the helix, are removed from steric interference with polypeptide backbone



Each peptide carbonyl is hydrogen bonded to the peptide N-H group 4 residues farther up the chain



All H bonds lie parallel to the helix axis and all the carbonyl groups are pointing in one direction along the helix axis while the N-H groups are pointing in the opposite direction

What is helix capping?

Providing H-bonds partners for the otherwise bare N-H and C=O groups and folding other parts of the protein to foster hydrophobic contacts with exposed non-polar residues at the helix termini

What are beta-pleated sheets?

Not planar


 


Important feature of protein secondary structure


 


Formed from extended chains of amino acids, bonded to each other through alternating intermolecule H-bonds


 


Every second aa is hydrophobic making one side ...

Not planar



Important feature of protein secondary structure



Formed from extended chains of amino acids, bonded to each other through alternating intermolecule H-bonds



Every second aa is hydrophobic making one side hydrophobic and other hydrophilic



Forms because of local, cooperative formation of hydrogen bonds



Pleated sheet can exist in both parallel and antiparallel forms



3.0 residues per translation

What is a parallel beta-pleated sheet?

Adjacent chains run in same direction



Tend to be more regular than antiparallel



Typically large structures; those composed of less than 5 strands are rare



Distribute hydrophobic side chains on both sides of sheet

What is an antiparallel beta-pleated sheet?

Adjacent strands run in opposite direction 


 


Can contain as few as 2 strands 


 


Usually arranged with all their hydrophobic residues on one side of the sheet

Adjacent strands run in opposite direction



Can contain as few as 2 strands



Usually arranged with all their hydrophobic residues on one side of the sheet

What are beta turns?

Designates the 4-residue segment through which the protein chain turns 180 degrees (fold back on itself, usually surface of protein)


 


AKA Reverse turns


 


Can link two beta strands, helices or strand to helix


 


Peptide c...

Designates the 4-residue segment through which the protein chain turns 180 degrees (fold back on itself, usually surface of protein)



AKA Reverse turns



Can link two beta strands, helices or strand to helix



Peptide chain forms a tight loop with carbonyl oxygen of 1 residue hydrogen bonded with the amide proton of the residue 3 position down the chain



Because it lacks a side chain, glycine is more steric the most adaptable of the amino acids and it accommodates conveniently to other steric constraints in the beta-turn



Proline, however, has a cyclic structure and a fixed phi angel, so, to some extent, it forces the formation of a beta-turn

What are phi/psi/omega/chi angels?

Dictate the secondary protein structures  


(alpha helix/beta sheets), thus  those angles define secondary structures 


 


The angle about the C alpha - N bond is denoted by the Greek letter phi and that about the C alpha - C is denot...

Dictate the secondary protein structures


(alpha helix/beta sheets), thus those angles define secondary structures



The angle about the C alpha - N bond is denoted by the Greek letter phi and that about the C alpha - C is denoted by psi



Omega is around the C-N peptide bond



Chi describes the side chain rotation

What are protein backbone angles?

Dihedral angle at each backbone bond determines the actual three-dimensional progress of the protein chain


 


Helices and sheets have characteristic backbone angles

Dihedral angle at each backbone bond determines the actual three-dimensional progress of the protein chain



Helices and sheets have characteristic backbone angles

What is the Ramachandran Plot?

Plotting group of proteins by phi and psi angles

Plotting group of proteins by phi and psi angles

What is Poly-proline helix?

put a bunch of Pro together to form a helix 


 


Pro doesn't have protons, thus Pro cannot  


form H-bond 


 


it has carboxyl group which acts as a proton  


acceptor BUT not a proton donor 


 


thus Pro can't be...

Put a bunch of Pro together to form a helix



Pro doesn't have protons, thus Pro cannot form H-bond



It has carboxyl group which acts as a proton acceptor BUT not a proton donor



Thus Pro can't be a H-bond donor, but it can work as a H-bond acceptor



When you put bunch of Pro's together, there's no NH groups, so it's not H-bonded at all



Polyprolinehelix has NO H-bonds due to its conformational restriction of Pro residue

Describe the fibrous protein, silk:

Most common residue is Gly



Has proton, conformational flexibility, can be in turns, alpha helix, beta sheets



Repeating unit of:


- G - S - G - A - G - A -


which is conducive to beta-pleated sheets



Inter-chain H-bonds are formed while side chains are above and below the plane of H-bond network



Spider silk (a form of keratin) is synthesized in special glands in the spider's abdomen



Strong and elastic



Dragline silk (from which the spider hangs) has a tensile strength of 200000 psi (pounds per square inch)



Small residue Gly allows tight packing



Hydrogen bond network link beta-strands



Fibers strong: resistance to tension is borne by covalent bonds

Describe the fibrous protein wool:

Mostly Glu + Gln



Is a keratins



Much lower in Gly vs silk, fairly even distribution of helix-forming residues (Ala, Glu)



7 amino acid heptad repeat



Cystine crosslinks



Can be stretched to twice its length



In stretched state, intra-helix H-bonds may be broken, and inter-strand H-bonds may form: via an alpha-helix to beta-sheet transition (structure NOT static, changes depends on humidity)



Due to sulfur content, disulfide crosslinks constitute the main restoring force

Describe the fibrous protein keratins:

Highly-complex macromolecular structure: each single alpha-helix is twisted with others into a "protofibril"


 


The alpha helices are bundled/packed together into protofibril which packs together to form microfilbril with 9-11 protofibr...

Highly-complex macromolecular structure: each single alpha-helix is twisted with others into a "protofibril"



The alpha helices are bundled/packed together into protofibril which packs together to form microfilbril with 9-11 protofibrils, to form a fiber resembling rope

Describe the fibrous protein collagen

High in Gly


 


Lots of Pro and modified version of Pro, hydroxy-proline 


 


can form turns and bents 


 


put bunch of proline together to form polyproline helix (no H-bonds to stabilize it) 


 


About 1/3 of ...

High in Gly



Lots of Pro and modified version of Pro, hydroxy-proline



Can form turns and bents



Put bunch of proline together to form polyproline helix (no H-bonds to stabilize it)



About 1/3 of our protein is collagen



Universal occurrence



Rigid, inert material, resistant to stretching



Used for connective tissue, skin, tendons



Collagen contains large proportions of glycine and proline and much of its structure is composed of (Gly-x-Pro) repeating units where x is any amino acid



Three unusual modified amino acids are also found in collagen:


4-hydroxyproline (Hyp)


3-hydroxyproline


5-hydroxylysine (Hyl)

What is the structure, short form and properties of Hydroxyproline

Hyp


 


OH group used to form H-bond cross links between collagen strands


 


Enzyme proline hydroxylase adds -OH group to proline side chain in pro-collagen protein


 


Thus no CODON for Hyp since it can only be made by the...

Hyp



OH group used to form H-bond cross links between collagen strands



Enzyme proline hydroxylase adds -OH group to proline side chain in pro-collagen protein



Thus no CODON for Hyp since it can only be made by the enzyme, proline hydroxylase



more hydrophilic because it has H-bond donor (the OH group is present in Hyp not Pro)



Hyp likes to interact with water

What is basic coiled-coil structure of collagen?

Three left-handed single-chain helices wrap around one another with a right-handed twist

Three left-handed single-chain helices wrap around one another with a right-handed twist

What are 3 fibers inside cells that make up cytoskeleton and what is cytoskeleton?

Actin


 


Microfilaments


 


Intermediate filaments


 


they are long rod-like structures 


 


vesicles are carried onto cytoskeletons  


 


Help move vesicles around (from ER togolgi) 


 


Motor prot...

Actin



Microfilaments



Intermediate filaments



they are long rod-like structures



vesicles are carried onto cytoskeletons



Help move vesicles around (from ER togolgi)



Motor proteins usually use ATP to move vesicles along the cytoskeleton

What is G-actin?

Single actin subunit


 


Single polypeptide (monomer) 


 


G for globular protein 


 


Actin is important in forming long fibers 

Single actin subunit



Single polypeptide (monomer)



G for globular protein



Actin is important in forming long fibers

What is F-actin?

F for filament 


 


We can shrink/expand actin filaments by adding/removing single actin subunits


 


Involved in cell motion


 


Actin filament is added to one end to make longer or fall off another end 

F for filament



We can shrink/expand actin filaments by adding/removing single actin subunits



Involved in cell motion



Actin filament is added to one end to make longer or fall off another end

What are globular protiens?

Have folded, roughly spherical structures



With right phi/psi angles, it can form alpha helices and beta strands



Many are enzymes, with active sites embedded in the protein core



Consists largely of secondary structure elements, including helices, sheets and turns



Polypeptide chain is compactly folded so that hydrophobic amino acid side chains are in the interior of the molecule and the hydrophilic side chains are on outside exposed to solvent, water



Usually very soluble in aqueous solutions



Globular proteins exist in an enormous variety of 3-dimensional structures but nearly all contain substantial amounts of alpha-helices and beta-sheets folded into a compact structure that is stabilized by both polar and non-polar group



Space between the helices and sheets in protein interior is filled efficiently and tightly with mostly hydrophobic amind acid side chains

What is the globular protein: myoglobin?

Richest source of it: muscles of aquatic vertebrates like whales.


 


First protein experimented for X-ray crystallography in 1958


 


'Box' for a heme group: heme binds oxygen, protein stores it until required for metabolic oxidatio...

Richest source of it: muscles of aquatic vertebrates like whales.



First protein experimented for X-ray crystallography in 1958



'Box' for a heme group: heme binds oxygen, protein stores it until required for metabolic oxidation



153 amino acids: 121/153 in alpha helices



8 connect helix segments connected by turns and loops



Strongly hydrophobic core



Helices fold via beta-turns: several turns contain Pro or Gly residues



The glu for the oxygen binding site is the His side chains



2 His residue shelp stabilize heme molecule in a very specific position

What is the globular protein: ribonuclease A?

Chain-cutting enzyme, cleaves the polyribonucleotide chains of RNA but not DNA


 


Taco-shaped molecule with active site in crevice running across the molecule


 


Has significant segments of beta-sheet and alpha-helix


 


Fou...

Chain-cutting enzyme, cleaves the polyribonucleotide chains of RNA but not DNA



Taco-shaped molecule with active site in crevice running across the molecule



Has significant segments of beta-sheet and alpha-helix



Four disulfide bonds to help stabilize the protein once outside the cell



Active site located by binding inhibitor which includes His 12, His 119 and Lys 41



Antiparallel structure



His119 and His12 right beside each other => critical for their activity

What is the mechanism of ribonuclease?

1) N on His12 has on proton, thus acts as nucleophile and pulls H+ from ribose hydroxyl group 


 


2) the O has 2 e- now since deprotonated and forms H-bond with phosphate 


 


3) now this O (from phosphate group) takes proton from...

1) N on His12 has on proton, thus acts as nucleophile and pulls H+ from ribose hydroxyl group



2) the O has 2 e- now since deprotonated and forms H-bond with phosphate



3) now this O (from phosphate group) takes proton from His119 and His119 becomes deprotonated



Lysine-41 is not directly involved in this mechanism, but it is there to stabilize the intermediate state



NOTE: DNA does not have OH at carbon 2 (unlike RNA's ribose sugar), thus rxn with deoxyribose sugar wouldn't work

What are membrane proteins?

Are responsible for key cellular processes



Functions: transporters, receptors, enzymes, cellular morphology



Bilayer maintains native conformation of proteins



Have to be compatible with hydrophobic phase of lipid bilayer

What are multi-spanning proteins: transporters?

Carry out exchange of substrates in and out of the cell across the plasma membrane



Channels and pumps



Energy (ATP) required for pumps



Electro-chemical gradient

Which transports do not require energy?

Proteins which use gradients across the membrane of the substance they transport



Example) Bind 3 anion transport of red cells, the glucose carrier protein

What is the uracil transporter?

Found in bacteria 


 


Almost 100% alpha helix 


 


Dynamic as it open and closed, bringing uracil in and out 


 


14 transmembrane protein sections  


 


Uracil is bound in middle, H exists in number of states: 
...

Found in bacteria



Almost 100% alpha helix



Dynamic as it open and closed, bringing uracil in and out



14 transmembrane protein sections



Uracil is bound in middle, H exists in number of states:



Closes up and releases uracil on other side, then opens up again and repeats this process



Recall: membrane proteins are highly helical

What are receptor proteins?

Receptors transmit signals across membranes (nothing is transported)



Ligand-activation fairly general mechanism (binding outside the cell induces signal)



Receptors may be single-spanning (insulin receptor) or multi-spanning (G-protein-coupled receptors)

What are insulin receptor signals?

Bind insulin -> change conformation of protein -> transmit info -> activate the kinase and it puts phosphate groups on tyrosine 


 


Made of 4 subunits 


 


Linked together by disulphide bonds 

Bind insulin -> change conformation of protein -> transmit info -> activate the kinase and it puts phosphate groups on tyrosine



Made of 4 subunits



Linked together by disulphide bonds

What is Anfinsen's experiment?

Anfinsen found that reduced RNase (using beta-mercaptoethanol) proceeds to completion only when the protein is partially unfolded by denaturing agents such as urea or guanidine hydrochloride


 


Anfinsen discovered that removing themercapto...

Anfinsen found that reduced RNase (using beta-mercaptoethanol) proceeds to completion only when the protein is partially unfolded by denaturing agents such as urea or guanidine hydrochloride



Anfinsen discovered that removing themercaptoethanl but not the urea restored only 1% of enzyme activity



If anfinsen removed mercaptoethanol and urea at the same time, the polypeptide was able to fold into its native structure, the correct set of 4 disulfides re-formed and full enzyme activity was recovered



This experiment demonstrated that the information needed for protien folding resided entirely within the amino acid sequence of the protein itself



The urea in this situation was able to form H-bonds with protein backbone.



Takes H-bond away from alpha helix that has H's, lets it bind with itself



Loss of structural order in these complex macromolecules (denaturation) is accompanied by loss of function



After dialysis to remove small molecules (urea and beta-mercaptoethanol), enzyme slowly regained activity



Sulfyhydral groups become re-oxidized by air to form the refolded, catalytically-active form



Results led to main conclusion that primary sequence determines secondary and tertiary structure



When RNase was refolded in 8M urea by oxidation and then dialyzed to remove urea, only 1% enzymatic activity was obtained.


-wrong disulfide pairing occured

How does denaturants work?

By disrupting (competing for) non-covalent interctions



Danaturation over a very small temperature range such as this evidence of a two-state transition between the native and the unfolded states of the protein and this implies that unfolding is an all-or-none process



When weak forces are disrupted in one part of the protein, the entire structure breaks down



Danaturation leads to loss of protein structure and function

What is dialysis?

If solution of proteins is separated from bathing solution by a semipermeable membrane, small molecules and ions can pass through the semipermeable membrane to equilibrate between the protein solution and bathing solution 


 


Method is us...

If solution of proteins is separated from bathing solution by a semipermeable membrane, small molecules and ions can pass through the semipermeable membrane to equilibrate between the protein solution and bathing solution



Method is useful for removing small molecules from macromolecular solutions or for altering the composition of protein-containing solution

How would you renature a ribonuclease?

Once you have denatured reduced ribonuclease



Dialysis to remove urea and beta-mercaptoethanol



Air oxidation of the sulhydryl groups in reduced ribonuclease to reform the disfulphide bridges



Then you will have native ribonuclease

What is native state of proteins?

Native state (with all interactions) is only marginally stable relative to the unfolded (denatured) state



Native state posses considerable "kinetic stability" because it can unfold only by passing through a high-energy transition state in which most native interactions are disrupted simultaneously



If proteins are in native conformation, last thing you want to not randomly just fly open (spontaneous denature)



Only way to unfold you must go through energy transition like losing part of helix, losing hydrogen bonds or van der walls

What is the cardboard box model?

In unfolded state, the "flaps" of the protein are free to move independently


 


entropy is lost during folding because gone from free moving state to a very restricted state (lose free of motion but you're gaining all non-covalent and cov...

In unfolded state, the "flaps" of the protein are free to move independently



entropy is lost during folding because gone from free moving state to a very restricted state (lose free of motion but you're gaining all non-covalent and covalent interactions to stabilize protein => thus balances the entropy and overall stabilization



Large energy barrier must be surmounted to convert from "almost folded" to "fully folded"



This guarantees that proteins do not denature too easily

What are isozymes:

Structural varients



These structure don't have negative effects, sometimes they might have positive effect



These variations results in the population (what makes everyone different)

What are restriction sites and enzymes?

Start with gene of interest within double stranded DNA


 


"A" and "B" are appropriate flanking endonuclease sites


 


Restriction endonucleases are enzymes found in procaryotic cells


 


Many restriction endonucleases recogni...

Start with gene of interest within double stranded DNA



"A" and "B" are appropriate flanking endonuclease sites



Restriction endonucleases are enzymes found in procaryotic cells



Many restriction endonucleases recognize 'palindromic' sites in double-stranded DNA



Such sites read the same sequence left-to-right and right-to-left

What is insertion of synthetic oligos?

Many genes contain wild type restriction sites: you choose the endonucleases based on palindromic sequence



Cleave the genes of interest at the restriction sites



Synthesize and purify the desired oligonucleotide (replacement therapy)



Insert the oligo into the chain enzymatically using "DNA ligases"

What is Thioredoxin?

You would add another protein sequence that E.coli loves, allowing your protein of interest to be expressed 


 


Essentially, you're using this to help express your protein of interest 


 


It acts as carrier and helps make your pr...

You would add another protein sequence that E.coli loves, allowing your protein of interest to be expressed



Essentially, you're using this to help express your protein of interest



It acts as carrier and helps make your protein soluble



Once you have the fusion protein expressed, you can cleave off the tag (since you're not trying to study it)



What are His-tags?

6-10 His residues for affinity chromatopgraphy


 


His-tags are small sequences that codes for many His residues (6-10) in a row 


 


His binds to metals (Ni and Co) in proteins 


 


This tag on the protein will bind to a re...

6-10 His residues for affinity chromatopgraphy



His-tags are small sequences that codes for many His residues (6-10) in a row



His binds to metals (Ni and Co) in proteins



This tag on the protein will bind to a resin that has Ni on it



Any protein that has His-tag on it, will bind to the resin



Proteins that don't have His-tag will not bind to resin



Ni will bind to proteins with His-tag, then you wash the proteins with imidazole to separate your protein of interest away from other proteins



Put at ends, not middle, because it can interfere with folding



Nickle affinity columns recognize His-tags and bind this protein exclusively. After other proteins are eluted, imidazole is used to elute protein of interest

What are WB-tags?

Western blot antibody tag


 


It is used for detection (to detect your protein) 


 


It uses antibody to recognize this particular tag

Western blot antibody tag



It is used for detection (to detect your protein)



It uses antibody to recognize this particular tag

What is protein purification?

Fusion (or carrier) proteins are often contained in expression vectors, which aid in solubilization of expressed proteins



One-step enzymatic cleavage is used at final steps to cleave off fusion protein



Concurrent with characterization, necessary to demonstrate that you have correct protein



Should appear as an extra band at the expected molecular weight



Use 'Western blot' with antibody to fusion protein to confirm identity

Why are plasmids useful in cloning genes?

Naturally occuring extra-chromosomal DNA


 


circular dsDNA (ds = double stranded)


 


Can be cleaved  by restriction enzymes, leaving sticky ends


 


Can be constructed by linking new DNA fragments to sticky ends of plasmid
...

Naturally occuring extra-chromosomal DNA



circular dsDNA (ds = double stranded)



Can be cleaved by restriction enzymes, leaving sticky ends



Can be constructed by linking new DNA fragments to sticky ends of plasmid



These recombinant molecules can be autonomously replicated and propagated



Cloning vectors are plasmids that can be modified to carry new genes



Plasmids are useful as cloning vectors as they have:



Replication (origin of replication, ori)


• ori allows DNAP to bind in order to allow it to make more copies of plasmid DNA



Selectable marker (antibiotic resistance gene)


• have TT of E.coli and you add DNA into it, the DNA will only go into SOME of the E.coli, not all


• you only want the E.coli's with your gene


• this gene will allow the bacteria to resist the antibiotic


• if the E.coli has the plasmid and it makes lots of it, it will make proteins that will resist to antibiotic


• you would take the E.coli and add antibiotic


• the only E.colis that will survive are the ones with your plasmid in it


• those bacterial cells will be antibiotic resistant because you put the antibiotic resistance gene into the plasmid (hence these are the cells that will express your protein)



Cloning site (site where insertion of foreign DNA will not disrupt replication or inactivate essential markers)

What are chimeric plasmids?

After cleave of a plasmid with restriction enzyme, a foreign DNA fragment can be inserted



Ends of the plasmid/fragment are closed to form a "recombinant plasmid"



Plasmid can replication when placed in suitable bacterial host if you have an ori

What is In Vitro mutagensis?

One method of PCR-based site-directed mutagenesis


 


1) Template DNA strands are separate and amplified by PCR


 


2) Following many cycles of PCR, the DNA product can be used to transform E. coli cells


 


3) The plasmid DNA...

One method of PCR-based site-directed mutagenesis



1) Template DNA strands are separate and amplified by PCR



2) Following many cycles of PCR, the DNA product can be used to transform E. coli cells



3) The plasmid DNA can be isolated and screened for the presence of the unique restriction site by restriction endonuclease cleavage

What are expression vectors?

You want to make the protein  


 


You engineering the DNA where a site that can be recognized and makes a lot of mRNA

You want to make the protein



You engineering the DNA where a site that can be recognized and makes a lot of mRNA

What is SDS gel electrophoresis?

SDS-PAGE: sodium dodecyl sulfate - polyacrylamide gel electrophoresis (denaturing detergent - meaning the protein loses its activity)


 


SDS: denaturing detergent and dissolves membranes and solubilizes proteins


 


Polyacrylamide: ...

SDS-PAGE: sodium dodecyl sulfate - polyacrylamide gel electrophoresis (denaturing detergent - meaning the protein loses its activity)



SDS: denaturing detergent and dissolves membranes and solubilizes proteins



Polyacrylamide: gel matrix to separate proteins (If protein small, they go through mesh easily and migrate faster)



Number of SDS molecules bound by a polypeptide is proportional to the length (number of amino acid residues) of the polypeptide



Each dedecyl sulfate contributes 2 negative charges



SDS-PAGE is usually run in the presence of sulfhydryl -reducing agents such as Beta -mercaptoethanol so that any disulfide links between polypeptide chains are broken



Often used to determine molecular weight of a protein

What are the lanes of an expressed SDS-PAGE gels of expressed proteins?

Lane 1: The lysate from E. coli - contains all proteins


 


Lane 2: The "flow-through" from the Ni-affinity column


 


Lane 3: The purified expressed (His-tagged) fusion protein eluted from column with 400 mM imidazole


 


Lan...

Lane 1: The lysate from E. coli - contains all proteins



Lane 2: The "flow-through" from the Ni-affinity column



Lane 3: The purified expressed (His-tagged) fusion protein eluted from column with 400 mM imidazole



Lane 1', 2', and 3': Same as Lane 1, 2, and 3, but blotted with an anti-fusion protein antibody. Only the fusion protein appears

What is an Western (Immuno) Blot?

Are used to purify or detect protein


 


Uses an antibody which is recognizing something particle like a his-tag


 


Has detection signal that produces fluorescent compounds


 


Has a primary antibody that recognizies the His-...

Are used to purify or detect protein



Uses an antibody which is recognizing something particle like a his-tag



Has detection signal that produces fluorescent compounds



Has a primary antibody that recognizies the His-tag and binds to it

What is subtilisin?

It is an endopeptidase, MW 27500 (275 residues)


 


Secret in large amounts from many Bacillus species


 


X-ray crystal structure determines there was 2 domains in this protein


 


Its active site features a charge-relay netw...

It is an endopeptidase, MW 27500 (275 residues)



Secret in large amounts from many Bacillus species



X-ray crystal structure determines there was 2 domains in this protein



Its active site features a charge-relay network involving Asp-32, His-64 and active site Ser-221



For the charge-relay system of subtilisin; Asp-32 carboxylate side chain H-bonds to N-bonded proton on His-64 imidazole ring



The other N-atom on His-64 H-bonds to the other O-H proton of Ser-221



Ser-221 becomes 'activated' by virtue of charge-separation of O-H, and its O-atom becomes more nucleophilic



Ser-221 O-atom attacks incoming substrate, assisted by a neighboring carboxyamide side chain of Asn-155



Substrate is susceptible peptide bond bound into the active site clef



This action causes the substrate peptide bond be cleave (needs water for this mechanism to work)



You can do mutagenesis of subtilisin to:



-Determine residues involved in catalysis (mutate Ser, His, Asp and Asn to see what happens)



- Also to enhance enzyme stability (can only cleave certain peptides)



-Obtain details of substrate specificity



-Determining effect of disulfide bonds on secretion, structure and stability

What is the catalytic mechanism of serine proteases?

Substrate coming in and binds to active site 


 


1) His will take proton offSer 


 


2) now you have intermediate, very unstable, electrons goes back to C and bond between N-C is broken 


 


3) now take H+ off imidazole, t...

Substrate coming in and binds to active site



1) His will take proton offSer



2) now you have intermediate, very unstable, electrons goes back to C and bond between N-C is broken



3) now take H+ off imidazole, the other part of peptide chain is covalently attached to the enzyme (acyl-intermediate)



4) since it was onlybindedby weak H-bond, it floats away



5) water must be added for hydrolysis reaction, the N on the ring takes H from water, and the OH will bind to the group that covalently bonded to enzyme



6) the Asp will come in todistabilizethis structure



7) bond betweenSerand C will be broken



8)Serhas proton back from water

What is cassette mutagensis of subtilisin?

Method: choose a codon in the gene and perform 'randomized mutagenesis' (not site directed, you don't know which codon might work)


 


Use 25% each of nucleotides A, C, T, G at each of three codon bases during oligonucleotide syntehsis. By ...

Method: choose a codon in the gene and perform 'randomized mutagenesis' (not site directed, you don't know which codon might work)



Use 25% each of nucleotides A, C, T, G at each of three codon bases during oligonucleotide syntehsis. By this method, any three-base codon can arise



Goal: engineering of oxidative resistance.



Substilisin known to be stable to denaturants, but highly susceptible to chemcial oxidation



Peptide mapping studies showed that oxidizig Met-222 (next to catalytic Ser-221) results in 90% loss of enzyme activity.



Methionine Oxidation can be done by oxidization of methionine to methionine sulfoxide to methionine sulfone



Met-222 is conserved residue in all subtilisins, suggesting funcitonal role



This is proved by fact that the enzyme is easily inactivated by oxidizing Met-222


What happens if you try to engineering disulfide bonds into subtilisin?

The enzyme has no Cys and no S-S bonds


 


Engineering problem: potential S-S bond must be chosen to conform geometrically to naturally-occuring S-S bonds, and not cause significant main chain rearrangement 


 


Cys must be placed ...

The enzyme has no Cys and no S-S bonds



Engineering problem: potential S-S bond must be chosen to conform geometrically to naturally-occuring S-S bonds, and not cause significant main chain rearrangement



Cys must be placed in proper place otherwise protein would not fold properly and not function



Ser-24 to Cys-24 or Ser-87 to Cys-87 (-CH2OH replaced by -CH2SH)



Both Ser residues in wild type subtilisin are located on back side of molecule distant from active site (24 angstroms away)



Results:



-Double Cys-Cys mutant was successfully expressed and isolated



-Secreted and processed normally by B. subtilis (novel engineering feat)



-Had the same specific activity as wild type enzyme



-X-ray confirmed presence of S-S bond, negligible alteration in protein structure



Studies of thermostability of s-S subtilisin were complicated by autolysis: the enzyme begins to digest itself (as substrate) as the temperature is raised



Rate of autoproteolytic inactivation increased as heating increases



Results showed no basic different between S-S and wild type subtilisin, either in specific activity, pH maximum for catalysis or autoproteolytic inactivation (didn't stabilize any more than the wild type)



Some secreted serine proteases have several S-S bonds, some have none

How would you predict a protein structure?

Minimum energy calculations can locate low energy regions for protein backbone and side chain rotational angles.



phi/psi angles will dictate whether that sequence will form alpha helix or beta strands



These calculations 'predict' the existence of favoured secondary structures



big residues like tryptophan, have big side chains => rotational angles are restricted => inside proteins it would be even more restricted



the linear arrangement allows to fold into alpha helix, allowing them to pack the side chains on surface



in 3D structure, the AA in distance will interact with those side chains



beta strands from other regions can interact



you're only predicting the structures, it may actually turn out differently



The alpha-helix and beta-sheet therefore involve local regions in which each residue repeats stereochemically-desirable angels



These procedures have led to the Ramachandran plots



But these calculations give information only about "short-range interactions"



Thus, protein tertiary structure cannot be predicted on this basis alone



At least two other factors must be considered:



1) Environment:



-Must be taken into account; most calculations are done in a vacuum: small energy increments gained or lost at hundreds of sites contribute to folding



-The E to unfold a protein is very low (5-10kcal which is the energy of one H-bond) because proteins are dynamic and have to move and work which means you cannot have a very stable elements



2) Long range protein/protein interactions must be considered to obtain the overall protein structure



-Especially pertinent to formation of inter-molecularly H-bonded structures such as beta-sheets (from sequentially) distant segments of a protein)



- sometimes the last strands of beta sheets will interact with each other to form beta barrels



- want to predict if these last strands interact or not


What are Chou-Fasman rules?

Conformations of given residue was determined by its psi/phi angels



Consecutive sequence of 4 or more helical residues are 'helix' (to make an alpha helix, each turn is 4 AA)



Consecutive sequences of 3 or more beta-sheet residues are "beta-sheet"



Residues not in either region are assigned "random" independent of phi/psi angels



Leu is good alpha helix former



If Leu in sequence, and it's onlyLeu, then it won't be alpha helix



If you have 4 Leu in a row, you're likely to have alpha helix



2-3 residues minimum for beta strand



3 great beta strand formers in a row, likely to be a beta strand

What are parameter designations?

Propensity (P_) alpha, beta and t refers to values for individual amino acids


 


To obtain average values, add up individual values and divide by number of residues

Propensity (P_) alpha, beta and t refers to values for individual amino acids



To obtain average values, add up individual values and divide by number of residues

What was Chou-Fasman results?

Conformational parameters (P-valueS) were obtained from % occurrences of each residue in each structural form, compared to the overall occurence of that form



Thus is if all Ala residues in the 30 proteins are considered, and 45% of them are in helical regions, then: Ala P(alpha) = 0.45/0.38 = 1.18



1 means no real presence



1.18 means rich in presence, higher than 1 is alpha helix



P-values were developed similarly for the three types of secondary structure:



P-alpha = propensity of a given residue to be helical



P-beta = propensity of given residue to be beta-sheet



P-t = propensity of given residue to be in a beta-turn



Val and Ile are great beta former and are beta branched (beta branched are great Beta formers)



Reason why beta branch not good in Alpha is because they are large and makes it hard to make it compact



Glu worst beta former despite being best alpha former



What are the top helix forming residues?

Glu, Met, Ala, Leu, Lys



Cannot have 4 Glu next to each other as Glu is negative and they repel each other unless it becomes protonated which means it cannot only form a perfect alpha helix if it is lower than it's pKa



These are hydrophobic



Negative charge repels each other (cannot form helix with 4Glu in a row) unless you add proton to neutralize it



Lower pH below the pKa of Glu, the negative charge are now neutralize

What are the best helix-breaking residues?

Gly, Pro, Asn, Tyr, Cys



Gly and Pro are most common turners



if Gly is in helix, it has a proton sticking out, it cannot be packed against helical structure



Cys has SH group, Tyr OH group are polar

What are the top beta-sheet forming residues?

Val, Ile, Tyr, Phe, Trp

What are the best beta-sheet breaking residues?

Glu, Asp, Pro, Gly, Lys



Glu has negative charge that can break the structure since it repels

What are the top beta-turn-occuring residues?

Asn, Gly (left handed helix at i+2), Pro (at i+1), Asp, Ser, Cys, Tyr

What are the least beta-turning occuring residues?

Ile, Val, Leu, Phe, Met

What is the difference between hemoglobin A vs S?

Sickle cell anime is a chronic hemolytic disease that is genetically transmitted.



Genetically transmitted means mutation in gene that is passed on from mother/farther to child



Hemoglobin A (HbA) is reversible oxygen carrier



Hemoglobin S (HbS) differs from HbA in one amino acid in the beta chain Glu-6-Val



This is a 'non-conservative' mutation: Glu is polar, charged amino acid, Val is highly non-polar aliphatic amino acid



This creates a hydrophobic patch on the surface of the protein



HbS forms long polymers that change the shape of red blood cells from normal to sickle



HbS tend to aggregate and forms long fibers, which deforms erythrocytes



There is a 'hydrophobic patch' on deoxyhemoglobin S that has a complementary binding site on another deoxyhemoglobin S molecule

Why does HbS aggregate?

Renaturation experiments have shown that information for folding into 2 and 3 structured is coded in the amino acid sequence



Combined knowledge of sequence/structure and prediction can provide insight into why HbS is a disease-causing form



Glu is strong helix former



Val is strong beta-sheet former



Going from Glu to Val, you change the propensity from a helix former to Val which would rather form a beta-sheet which would destabilize it and if this keeps happening, it would be destabilized even more and eventually form a beta sheet



This patch sits on the top of the surface and cannot escape which destabilize the alpha helix which is on the surface of the protein



It forms a helix most of the time but sometimes it might unfold and TRY to form a beta sheet

What is Glioblastoma?

Mutation in membrane protein neu/erbB2 proto-oncogene of mutation Val664 to Glu

Mutation in membrane protein neu/erbB2 proto-oncogene of mutation Val664 to Glu

What is the hormone: glucagon?

Opposite of insulin



29 residues, 'small' protein = easy to study



Secreted by the pancrease when blood sugar is low



Binds to receptor in liver cells to release glucose into the blood



Has N-terminal beta-strand and C-terminal helical regions



Spectroscopic measurements by circular dichroism have shown that glucagon changes structure as a function of concentration



At low concentrations, glucagon has C-terminal alpha helix (half beta, half alpha)



At high concentrations, glucagon is largely beta-structure (Mostly beta and eventually all beta and precipices out of solution and forms a gel)



Concentrated solutions of glucagon form a gel much like gelatin



CF analysis of glucagon results suggest a biological implication of glucagon segment may change conformation during its biological action



Storm formed is probably beta formed



Released form in blood, changes from all beta to half alpha, half beta



Bound to receptor form which shows helical conformation of peptide when bound to receptor because binding site of peptide is hydrophobic and becomes alpha helices becomes more hydrophobic so it can bind to binding site which has residues with poke out of the helical and fit perfectly to the receptor


What is gel filtration chromatography?

Chromatography column separates protein by size (molecular weight)


 


Column matrix (resin) consists of porous beads with varying pore size


 


Like a sponge with holes in it which small things can go in and get stuck


 


Cal...

Chromatography column separates protein by size (molecular weight)



Column matrix (resin) consists of porous beads with varying pore size



Like a sponge with holes in it which small things can go in and get stuck



Calibrate with protein of known size



smaller proteins are selectively trapped (and slowed)



Large proteins elute first

What is affinity chromatography

Chromatography columns separates proteins by selectively bidning specific proteins


 


Resin contains covalently bound substrate or ligand that recognizes one protein in the mixture and binds it with high affinity to the colum


 


Yo...

Chromatography columns separates proteins by selectively bidning specific proteins



Resin contains covalently bound substrate or ligand that recognizes one protein in the mixture and binds it with high affinity to the colum



You pick the ligand (Ni for His tag)



If you want insulin receptor, insulin would bind to column and if you add free insulin, it knocks it off the column and this would purify the insulin receptor



Selected protein molecules remain bound to the column, when unbound proteins are eluted through the column



Once separated, low molecular weight substrate or ligand is added to the column in excess and "competes" the bound protein off the column

What is HPLC?

High Pressure (Performance) Liquid Chromatography


 


Not best for proteins because hydrophobic are buried and will not show the hydrophobic patch but there are some proteins that does like HbS 


 


Contains hydropbic resin


 
...

High Pressure (Performance) Liquid Chromatography



Not best for proteins because hydrophobic are buried and will not show the hydrophobic patch but there are some proteins that does like HbS



Contains hydropbic resin



Separates peptides/proteins primarily by 'hydrophobicity'



More hydropbic compounds interact strong with the column, have longer 'retention times' on the colum

What is Ion Exchange Chromatography?

Separates amino acids or peptides mainly by positive or negative characters



Very useful to separate protein because charges are on surface on protein to combine as resin and get them off by adding a salt like KCl



Used in amino acid analysis to determine protein composition



Cation exchange resin has negative sites: attracts and tightly binds positively charged peptides/proteins



Anion exchange resin has positive sites: attracts and tightly binds negatively charged peptides/proteins

What is Edman degradation?

Chromophoric reagent combines with N-terminal amino acid, cleaves next peptide bond in the process


 


Creating a derivative and do this again 


 


Limited by length of sequence

Chromophoric reagent combines with N-terminal amino acid, cleaves next peptide bond in the process



Creating a derivative and do this again



Limited by length of sequence

What is carboxypeptidase?

Enzyme that cleaves amino acids one at a time from C(carboxyl)-terminus



Limited by length of sequence

What is Trypsin?

Enzyme cleaves to carboxyl side of basic residues (Lys, Arg)

What is Chymotrypsin?

Enzyme cleaves to carboxyl side of aromatic residues (Trp, Tyr, Phe)



Binding site in active site that recognizes aromatic residues of Trp, Tyr and Phe

What is Cyanogen bromide (CNBr)?

Chemically cleaves to the carboxyl side of Met residues

How can you use Mass Spectrometry (MS) to identify peptide fragments?

Protein (peptides) bombarded by high energy electron beams or laser beams


 


Ionized molecules created, which impact upon a charged plate: exact molecular weight detected


 


Charged = lose protons and become negatively charged and ...

Protein (peptides) bombarded by high energy electron beams or laser beams



Ionized molecules created, which impact upon a charged plate: exact molecular weight detected



Charged = lose protons and become negatively charged and mess up the protein and ionizing them into gas phase

What is X-ray crystallography?

(Some) proteins can form ordered crystals


 


Crystals placed in X-ray diffractometer produce diffraction patterns that can be interpreted in terms of atomic positions


 


Three-dimensional structures of proteins can be reconstructed...

(Some) proteins can form ordered crystals



Crystals placed in X-ray diffractometer produce diffraction patterns that can be interpreted in terms of atomic positions



Three-dimensional structures of proteins can be reconstructed at high resolution (2A), including backbone and side chains



Protein have hydrophilic surface that interacts with water, we want to use reagents that interacts with water in order to remove the water (ammonium sulphate)



Or force proteins in an ordered array to remove water



Membrane proteins are hard to crystallize because you must extract the proteins away from the lipid bilayer



The folding of the protein backbone can be specified preceisely using crystallographic data



The phi/psi angels of each residue on the alpha-carbon



Position (coordinates: x,y,z) of every atom is 3-dimensional space

What is NMR spectropscopy?

NMR = Nuclear magnetic resonance



In the presence of external amgnetic field, the nucleus (H proton) can exhibit more than one spin state (up/down)



Can move between these states by the absorption of electromagnetic radiation of specific frequency (energy)



The energy absorbed can be detected and from this information about the environment of the nucleus can be deduced



Very high field superconducting magnets (up to 800 MHz) provide spectra with great resolution and sensitivity



Protein structures can be solved with NMr

What are the advantages and disadvantages of x-ray and NMR?

Advantage: These are two most established high resolution techniques for deduction of protein structure



Disadvantages: X-ray proteins must be crystallized and for NMr proteins must be small



Large complexes can be visualized using electron microscopy (EM)

What is Infra-red spectropscopy?

Proteins contain vibrating, stretching and bending groups


 


In an infra-red beam, these motions lead to absorption of IR light as function of wavelength


 


Proteins have numerous C-H, N-H and C=O groups so these are most prominent...

Proteins contain vibrating, stretching and bending groups



In an infra-red beam, these motions lead to absorption of IR light as function of wavelength



Proteins have numerous C-H, N-H and C=O groups so these are most prominent in the Ir spectrum



Ir has the ability to detect and evaluate quantitatively H-bonding interactions



Band positions in the carbonyl amide region can distinguish helix and beta-sheet structures



Band positions correlate with "strength" of hydrogen bond

What are protein chromophores?

Operational definition: a chemical group that absorbs ultraviolet (UV) and/or visible light at characteristics wavelengths



Chromophore groups are usually contain conjugated double bonds



Amide carbonyls and aromatic rings are important chromophores in proteins

What is circular dichroism spectroscopy?

CD spectra record absorption of UV light, and also take advantage of asymmetry of proteins


 


Each alpha-carbon in each amino acid (except Gly) is asymmetric -L- vs D- configuation


 


Secondary structures are asymmetric - the twist...

CD spectra record absorption of UV light, and also take advantage of asymmetry of proteins



Each alpha-carbon in each amino acid (except Gly) is asymmetric -L- vs D- configuation



Secondary structures are asymmetric - the twist of a helix can be left or right handed



Proteins with asymmetric features can polarize light



Dissolve protein sample in water or suitable solvent



Place in small cuvette in UV beam



Polarize the UV beam



Spectrum records composite content of alpha-helix, beta-sheet and non-ordered structure in the protein

Which of the following represents an amide linkage?

Which of the following represents an amide linkage?

A

What is the corn rule?

With hydrogen in back:


 


If you spell corn clockwise with hydrogen back, that is D 


 


If you spell corn ccw with H back, that is L

With hydrogen in back:



If you spell corn clockwise with hydrogen back, that is D



If you spell corn ccw with H back, that is L