BCHM exam 2

How does heat denature proteins?

- Heat = increased kinetic energy of atoms. They move more and this kinetic energy overcomes the weak, non-covalent bonds within the protein. The protein often precipitates out due to hydrophobic group aggregation. - as temp increases, delta G increases from negative to positive so unfolding is favored - on graph of absorbance and temp (y,x respectively), the more folded proteins will be to the left and the more unfolded will be to the right bc aromatic aa residues that show absorbance are on inside when folded so less absorbance but when protein starts to unfold, the conjugated aa residues are now exposed and absorbance increase

why are there so many peaks in and ESI mass spec?

- bc the peaks have the same mass (m) but they have different charges (z), so mass to charge ratio is different--> appear as different peaks

would an ionic bond between two charged residues be stronger on the outside of a protein (exposed) or buried within the inside of a protein? why?

- depends on environment on surface of protein: - surrounded by water--> high dielectric constant - high dielectric constant = weaker ionic interaction - recall: strength = 1/ dielectric - so on outside, ionic bond would be weaker - H2O present could hydrate the ions, so more likely to break bond buried in protein: - would be within hydrophobic core with little water--> dielectric constant would be much lower, so teh bond would be much stronger

What aspects of protein folding contribute to positive entropy change (+ delta S)?

- hydrophobic effect: - water molecules surrounding the non-polar groups (the hydration shell) are ordered - BUT, they become disordered as more np hydrophobic groups pull closer to each other - this is bc there is less surface area of hydrophobic molecules as they aggregate together--> means fewer water molecules can be organized around them--> increase in entropy **its the increase in entropy of water molecules (not non-polar molecules)

what is the difference between using mass spec and x ray chrystallography?

- mass spec gives weight and amino acid sequence - x ray gives 3D structure

what are distal and proximal His?

- proximal His is a His residue weakly covalently bonded to the iron in the heme of Hb and Mb - distal His: the O2 that is bound to the iron in the heme is also bound to a His residue

kd (rate constant)

- rate constant for dissociation of protein to ligand

framework mechanism

- secondary structure forms first when folding a protein - then tertiary forms and hydrophobic groups are buried - part of old view

two methods for determining aa residue propensities to be in alpha helix

1. delta delta G values 2. counting the number of residues see in a structure

what is on the X axis of mass spec?

m/z - mass to charge ratio

how would you construct a Ramachandran plot?

1. look at each alpha carbon in backbone 2. determine the rotation of each phi (alpha C - NH) and each psi (alpha C and - COO) using surrounding bonds 3. plot each (phi, psi) pair like (x,y) on graph 4. the plot will have as many points as there are alpha carbons in the peptide (number of residues)

peptide prolyl cis-trans isomerase

- catalyzes interconversion of cis and trans isomers of proline peptide bonds

why does an alpha helix have a permanent dipole?

- every peptide in bond has a dipole bc of the resonance of the peptide bond (the lone pair on the N can move to create partial double bond character) - each peptide bond dipole lines up to create one larger net dipole through the helix - the positive end is at the N terminus (usually the bottom on diagram) - the negative end is the C terminus

quaternary structure

- highest level of structure (assembled subunits) - more than one chain and chains are associated non-covalently - protein-protein interaction due to: shape (puzzle pieces fit together) and chemical complementary (+/- charges)

what is the function of the matrix in MALDI mass spec?

- matrix absorbs energy from laser pulse of UV light - absorbed light causes matrix material to vaporize into gas phase allowing material to be analyzed by mass spec

why is heme bond to oxygen weakly covalent?

- so that the oxygen can hop on and off and isn't permanently stuck on

what is the most common non-covalent bond btn non-polar aa residues? describe the interaction.

Van der Waals - one electron cloud repels the other--> induces dipoles within the individual molecules - the dipoles can then form weak, attractive electrostatic bond - falls off with distance

in alpha helix, what number residue side chains will stick out on same side?

- # 1,4,7,10 (every three will be on same side) - bc helix repeats rotation every three turns (every 3.6 aa residues)

What do chaperones do?

- Chaperones are also called 'heat-shock proteins' and are present in all cells - The are responsible for the correct folding of proteins and help proteins reach their correct tertiary structure. - they prevent proteins from aggregating which allows them to have more time to fold properly - GroEL is HSP that can help misfolded proteins fold correctly

What can be concluded about the size and magnitude of the charges on eluted fractions that used DEAE chromatography (basically anion exchange)?

- DEAE is positive charge at pH 8--> meaning negative proteins would stick to the beads (positive charged beads) - fractions eluted first would have a weaker negative charge - fractions eluted last would have a larger negative charge bc they would stick better to the positively charged beads - if the fraction didn't stick at all, most like a positive charge because would repel and elute immediately

Describe X-ray crystallography

- DNA is bombarded with x-rays, this causes the pattern to be captured on film - limiting factor is obtaining a crystal structure of a protein 1. crystallize protein 2. use x ray beams--> diffraction pattern helps find the structure 3. the spots on diffraction pattern are constructive interference that indicate 2D distribution of protein which depends on how the electrons are distributed 4. the spots are where the electrons are - electron distribution is heavier near the main chain and surrounding atoms - get (x,y,z) coordinates and make the model--> see below 5. many images taken at different angles and then the structure is identified

why shouldn't too many np residues or polar residues be back to back in a helix?

- because of the rotation and how the side chains stick out - if have 4 right next to each other, some will be sticking out and some will be sticking in and that means at least one will be on the wrong side (hydrophobic vs hydrophilic) --> would cause destabilization of helx

Psi angle and bonds used to determine it

- between alpha C and C=O group of amino acid residue - use the C alpha - NH bond on one side and the C=O -NH bond on the other * put the C alpha in the middle, the C alpha-N bond in front facing down and see what the angle of the back bond is (the C=O -N bond)

Phi angle and bonds used to determine it

- between the N and the alpha Carbon - use the C alpha and C=O bond on one side and the NH-C=O bond on the other side * put the N- C=O in front of the alpha C with the C=O pointing down and see what angle the back bond is (C aplha - C=O)

What would you do to get a positively charged protein to stick to positively charged beads in column exchange chromatography?

- change the pH of the solution to have a higher isoelectric point for the protein--> this would give the protein a net negative charge which would allow it to stick to the positively charged beads

how does pH denature proteins?

- change the pKa of aa residues--> change the overall charge of the protein--> bread the H bonds--> denature - at low pH--> protonation of His (becomes positive) - at high pH--> deprotonation of Tryosine--> becomes negative - both cause destabilization (esp if they are buried!)

disulfide bonds

- covalent bonds that form btwn cysteine residues - usually found in proteins outside the cell (ie trypsin)--> the increased concentration of reducing agents inside the cell make disulfide bonds hard to form there - SH are oxidized and the two S bond together

protein disulfide isomerase

- enzyme which catalyzes the formation and shuffling of disulfide bonds - gets protein into native conformation

Kd (dissociation constant)

- equilibrium constant for the release of the ligand Kd = ([P][L])/[PL] = kd/ka - the lower the Kd value, the higher the affinity of ligand to protein

Where are the H bonds in an alpha helix?

- every 4th peptide bond - residue i to residue i + 4 - the O of the C=O to the H of the NH

describe alpha helix

- every peptide bond in the helix makes 2 H bonds (one to bond below and one to bond above)--> the H from the NH bonds to the O from the C=O - all are right handed - 3.6 aa residues for each helical turn (5.4 A) - side chains stick out and not really involved in forming of the alpha helix (so helixes can have many diff side chain combos) - side chains are essential for stability! - aa residues 1,4,7,10 will be polar and 2,3,5,6,8,9 will be np for amphipathic helix

what was the main thing the Anfinsen experiment determined?

- folded structure of a protein is determined by aa sequence of protein and no other factors were required for the protein to fold to that structure - but! doesnt say anything about pathway

structure and properties of folded proteins are determined by non-covalent interactions btwn______

- groups on a protein interacting with each other - groups on a protein and their interaction with water - water interacting with other water surrounding protein

Describe the race car analogy for protein folding.

- high points are unfolded intermediates at high energy ("mountains" on the funnel) - lower points are partially folded intermediates with less energy ("valleys") - different valley and mountains represent different paths encountered at different sets of intermediates (bc multiple pathways possible) - proteins that enter the funnel at different starting conformations (cities) encounter different intermediates (hills and valleys) en route to folding (destination city) - different routes to destination represent diff protein folding pathways and their intermediates

Why does delta S (of water molecules) increase when protein folds?

- hydrophobic effect - less surface area of np residues = less ordered water around them - entropy of water increases as np molecules aggregate

How would you make a polypeptide (a-helix) that would be stable in hydrophobic environment of a lipid membrane?

- hydrophobic environment--> do NOT use charged or np residues - use only np residues for most stable

in terms of "i", where are charge charge interactions (salt bridge or ionic bonds) likely to form in helix?

- i and i +1 - i and i+3 - i and i +4 - H bonds have to be a little closer that salt bridges - never i and i +2 bc that residue is like around the back of the helix

how would you organize an amphipathic peptide in a Beta sheet?

- in B sheets, the aa side chains will alternate btwn up and down - unlike helix where you have them grouped 1,4,7... this will be every other one - 1,3,5,7.. will be polar or np - 2,4,6,8.. will be the opposite

as dielectric constant increases (water), the ionic strength of a bond____ and the pka ____ as dielectric constant decreases (hydrophobic/ np solvent), the ionic strength of a bond____ and the pka ____

- increase Dielectric = decrease in ionic bond strength and increase in pka - decrease in Dielectric = increase in ionic bond strength and decrease in pka

protein domains

- individually folded areas of protein structure - can have different functions - part of tertiary structure and part of single polypeptide chain - not subunits

what non-covalent interactions are involved in stabilizing amyloid fibers composed of alpha-beta peptide? name specific groups

- ionic bonds btwn the Asp and Lys in the peptide - the individual B strands in the peptide fold back on themselves and the aa side chains interlock (connect) and form van der waals bonds

What does Beta-mercaptoethanol do (Anfinsen experiemnt)?

- it is a reducing agent used to disrupt disulfide bonds - breaks cysteine disulfide bonds by reducing them (S-S bonds) to thiols (-SH and -SH)

why is the + end of the helical dipole important at the N terminus

- it is important in enzymes and acts almost like a positive side chain

Describe size exclusion chromatography

- large fraction elutes faster - small elute later - small proteins can enter the pores/ channels in the beads which slows their elution - there is a large volume accessible to small proteins (more area where they can travel) and so more volume of buffer is needed to elute them out - large proteins need less volume of buffer because there is less space accessible to them so less buffer needed to flush them out

describe beta sheets

- main chain atoms all like in the plane of the screen - R groups and H off alpha carbons alternate pointing up and down - all R up will be dash or wedge and all R down will be opposite (same with H) - antiparallel: going in opposite directions - parallel: going in same direction - side chains can interact (non-covalent bonds)

Why do proteins denature at low pH (specifically pH less than/ equal to 5)? (hint: there is one specific amino acid to talk about)

- many proteins have His residues buried - His has pKa of 6--> above pH 6, His loses H+ and becomes neutral charge - below pH 6, His has + charge because still has it's H+ - the charged His on the interior of the protein causes instability! --> destabilize = denaturation - can't refold until pH increases and His gets protonated again and becomes neutral charge

How would mercap affect a protein Tm if the protein has disulfide bonds?

- mercap breaks disulfide bonds - break bonds = less stable = lower Tm

how do you calculate the molecular weight of a protein on an ESI mass spec?

- n = number of charges on ion - x = 1 and is ignored - adjacent peaks on spec are ions that differ by 1 charge - start by picking any two adjacent peaks - set value of peak to the left as the n+1 value and the peak to the right (bigger peak) as the n value 1. use two equations: m/z = (MW + nx)/n and (m/z)n+1 = (MW +(n+1)x)/(n+1) - find MW = equation for both and set both equal to each other - get this: [n*(m/z -1)] = [(n+1)*(m/z-1)] 2. fill in right m/z value (first m/z in equation above) with peak value farthest left and fill in left m/z value (second m/z in equation) with the adjacent peak value that is furthest right. 3. solve for n 4. plug n back into the two equations in step one using the correct m/z values and solve for MW 5. that will give you two values for MW--> take the average of the two and that give you the molecular weight!

Describe some effects that charged residues have on melting point (Tm) based on their location in the polypeptide.

- negative charges at the C terminus will have destabilizing effect --> lower Tm (bc the C term is negative charged and that is also the neg end of the helix dipole) - positive charges at the N terminus will have a destabilizing effect--> lower Tm (bc N term is pos charged and that is also the pos end of the helix dipole) - neg charges at N term will stabilize--> increase Tm - pos charges at C term will stabilize--> increase Tm - charged residues next to each other (unbonded to opposite charged residue) --> destabilize --> decrease Tm

Properties of 3D structure (where are different types of aa side chains located)

- non-polar aa side chains are on the inside (buried) *hydrophobic effect favors folding bc increase in delta S of water molecules *Van der waals btwn aa side chains inside protein favor folding (negative H bc bonds formed) - polar, charged aa are usually exposed on the surface *H and S stay bout the same as long as they are bonded to water or to each other *burying a charged protein that remains UNbonded is energetically unfavorable bc dehydrating (breaking the charged aa H bonded to water to put on the inside) is a positive delta G value *if buried but bonded (salt bridge), it is energetically favorable *breaking of charged aa bond to water is favorable bc S increase - polar, uncharged aa are mostly on surface (still want to make H bonds); can be both buried or exposed *outside prob H bonded to water *inside prob H bonded to other polar aa residues *H will stay the same if bonds broken reform (no net change) *S will increase when polar aa res break bonds with water to make bonds on inside with other polar aa residues--> water increases entropy

Describe the interactions between water and non-polar side chains in an unfolded protein. How do they change when protein folds? Do these interactions favor folding of the protein? Explain.

- np residues in unfolded are surrounded by a shell of water molecules (organized, lower entropy)--> when the np molecules come together in hydrophobic effect (folding), the water molecules become less organized and the entropy increases - the increase in entropy of the water molecules is favored in protein folding--> want positive delta S and negative delta H for folding - Van der Waals in Hydrophobic core gives negative H

what does Y represent?

- number of ligand binding sites Y = [L]/([L]+(1/Ka)) or Y = [L]/([L]+Kd) - in binding curve, Y answers the following question: of all ligand bind sites present in solution (aka total protein present), what fraction of those sites are actually occupied with ligand, making [PL] - ranges: 0 < Y < 1.0 - at low [L]total, most ligand binding sites are empty so Y = 0 - at high [L]total, most ligand sites are occupied--> [PL] aprrox = to [P] total

What happens to the number of points on a Ramachandran plot as the side residues get bigger?

- number of possible phi and psi conformations decrease because steric hinderance blocks angles from being able to form *less available conformations

where are non-polar residues found on an alpha-helix?

- on the inside (should be) - bc the hydrophobic effect encourages np residues to aggregate together to keep polar water out - burry themselves and make Van der Waals interactions with each other (np residues) to stabilize

where should charged amino acids be in alpha helix?

- on the outside of the structure for stabilization - positive can be near C term bc it has - charge - neg should be near N term bc it has + charge

What is the old view of protein fodling?

- one single pathway by which an unfolded protein would reach a folded state - intermediates are possible (partially folded)

How would a polar uncharged amino act in folded and unfolded protein- what bonds can it form and how does that contribute to entropy and enthalpy? Does it favor folding? (ie: Threonine, Serine, Asparagine, Glutamine)

- polar and uncharged in folded protein would form H bonds to water if exposed or with other aa side chains if exposed or buried - in unfolded, they would form H bonds with water surrounding it - delta H would be approx 0 bc if bonds are broken during folding, they will most likely be reformed by either making a new H bond with water or with other side chains--> little effect on protein folding - delta S would be positive bc the H bonds with water breaking during folding causes and increase in entropy of water--> would favor folding

describe helical capping

- polar and uncharged residues are found to stabilize helix when at ends - will destabilize more if in middle of helix - H bonding of side chain to N or C terminals (main chain-side chain interaction) - Asn, Ser, Thr (not super common in helix but can end cap)

Describe the interactions between water and polar (uncharged) amino acid side chains in the unfolded protein. How do the interactions change when protein folds? Do the interactions in the unfolded state favor folding? Explain.

- polar and uncharged side chains can form H bonds to surrounding water molecules in unfolded state - when the protein folds, the H bonds will either stay intact (if the residue will remain on the surface) or they can break and then the residues can form new H bonds with other side chains - of the number of bonds broken and formed is the same (no net change) then there will be no net change in enthalpy--> no large decrease in enthalpy means it probably doesn't affect protein folding that much - but--> the H bonds btwn the uncharged polar residues and water molecules breaking during folding can cause and increase in entropy of water molecules and that does favor protein folding

what were important conclusions drawn from identifying the structure of myoglobin?

- positioning of side chains reflect stability (like hydrophobic effect) - most polar groups on outside are hydrated - dense hydrophobic core common in globular proteins - van der waals inside with np residues stabilize protein - helped understand structure = function

describe heme

- prosthetic group - separate molecule but "permanently" bound-->. enables protein to perform function - iron that can bind to 6 things: binds to 4 N and one proximal His (weak covalent bond) and O2 via weak covalent bond - each N is part of 5 membered ring and is connected with one carbon (planar)

allosteric protein

- protein in which the binding of a ligand to one site affects the binding properties of another site on the same protein

what does it mean that proteins bind specifically to a ligand and how is that achieved?

- proteins bind tightly (with high affinity) to the biologically-relevant ligand - proteins bind much less rightly to other ligands - achieved by: chemical complementarity and shape complementarity

intrinsically disordered proteins

- proteins that are distinct from those of classical structure - could lack hydrophobic core or have high densities of Pro, Gly, charged aa - allows proteins to interact with many partners - can cause inhibition of other proteins - flexibility could cause them to wrap around other proteins

What happens to the number of points on a Ramachandran plot as the side residues get smaller? or you remove the C=O (double bond from peptide)

- removing the double bond from the carbonyl in the peptide bond means there is no more resonance structures available - no more resonance = no more delocalization of lone pair of electrons on N--> no partial double bond character of the carbonyl C - N bond in the peptide bond - no more double bond character = no more restricted rotation = MORE available conformations = MORE points on plot

how would you organize an amphipathic peptide in an alpha helix?

- residues 1,4,7,10... would be all non polar or polar - residues 2,3,5,6,8,9.. would all be the opposite! - you can do it either way (np-p-p-np OR p-np-np-p) but make sure charges are right - all charged residues would be on 1,4,7 ones pointing out! dont want them on the other numbers because have to consider their charge interactions bc they are right next to each other - reasoning: side chains that are three aa residues apart will point in same direction due to rotation

Where do proteins with disulfide bonds usually function?

- secreted outside of cell and function out of cell

what are the limitations of a psi bond?

- some psi angles will cause steric clash between the carbonyl and the other groups present in the side chain - the alpha C that is attached to the COO that makes the psi bond is the one with the side chains

what would happen to the entropy if the C=O in a peptide bond was replaced with a smaller, non-double bond, linkage? (ie: C=O replaced with CH2 linkage in polypeptide)

- the entropy of the smaller non-double bond linkage would be LESS (more negative) than the regular polypeptide with the double bond - bc the unfolded smaller one is more flexible (no double bond character)--> so can take on many more conformations --> the lack of double bond character allows more conformations to form bc rotation is not restricted! - so, there is a greater DECREASE in entropy as the single bond smaller polypeptide folds --> meaning the entropy (disorganization) is decreasing more bc it started with more possible options of conformations - so the polypeptide with the less possible conformations to be starting in (bc of the double bond) will have a smaller way to go to become more organized than the one with many possible starting conformations - *bc of the more possible conformations of the smaller no double bond polypeptide, it is LESS stable than the C=O regular peptide in the unfolded state, so its delta S will be more negative!

Describe the new view of protein folding.

- the funnel - unfolded protein can be in a large number of possible conformations (not just one possible starting point) - there are many pathways a protein can get to reach folded conformation--> means many possible intermediates - intermediates and pathways can have different levels of energy (indicated by the hills and valleys in the funnel) before reached it's lowest energy conformation state (the Native state) at bottom of the funnel - top lip of funnel (flat part) represents the protein starting at many different high energy unfolded conformations

Explain MS/MS spec

- the instrument has two mass specs that are linked together and two different but related mixtures are analyzed in each spec - this first mixture is of peptides that ionize as they pass through the nozzle and enter the first spec. these peptides are produced from original protein by being digested with a protease like trypsin - one peptide ion from first MS mix is allowed to enter the collision cell btwn the specs - cell is filled with an inert gas like argon - peptide ion collides with the gas and breaks into a mix of fragment ions--> peptide bonds break into "y" and "b" ions - both y an b ions enter MS chamber 2 where they are separated by their m/z and are detected

how does the pka of a charged residue change if unbound and in interior of protein (hydrophobic core)? what about exposed?

- the pka would decrease in buried hydrophobic environ bc the the dielectric constant is low--> lower dielectric constant = lower the pka--> the lower the pka, the stronger the acid--> the more likely it is to give the proton away! - the pka would increase in the hydrophilic environment bc the dielectric constant is high--> higher the constant = higher the pka--> higher the pka = weaker the acid--> weaker the acid, the less likely it is to give up the H!

what is proteomics?

- the study of the full protein set encoded by a genome - identifying every protein in a biological sample - done with chromatography and mass spec--> gives sequence of amino acids and then you check the database - goal: identify as many proteins as you can in a sample - ex: identify proteins in cancer and human cells --.> can lead to identification of what proteins are resistant to cancer drugs

What is the overall delta S of protein folding and unfolding equilibrium?

- the sum of: the more positive entropy (from the hydrophobic effect) plus the more negative entropy (from the folding of a more disorganized unfolding polypeptide to a more organized folded polypeptide)

What is the overall delta H of protein folding and unfolding equilibrium?

- the sum of: the negative Delta H for all bonds forming plus the of the positive Delta H for all bonds breaking during folding

What is Tm?

- the temperature at which 1/2 the protein is folded and the other 1/2 is unfolded (basically middle of slope on graph)

Why is RNase A less active when remove urea slowly and B-mercaptoethanol rapidly than compared to removing them both slowly? Explain in terms of the folding funnel mechanism.

- there are many paths that an unfolded protein can take to fold - proteins can also fold incorrectly, but can get stuck in a wrong conformation is it is low energy enough - If you remove mercap too slowing, the incorrectly folded proteins have a chance to unfold, then refold--> more likely to end up in correct structure - If remove the mercap fast, the disulfide bonds can form rapidly - recall: disulfide bonds are covalent and irreversible--> proteins more likely to be stuck in incorrect folded shape

why isn't proline in helix that often?

- there is no H on side chain to H bond bc of the 5 membered ring shape of the side chain - causes steric hinderance

Why was dialysis done in the Anfinsen experiment?

- to separate the denatured ribonuclease A proteins from the urea and mercaptoethanol - to test whether the denatured protein could fold back to native state

What aspects of protein folding contribute to a negative entropy change (- delta S)?

- unfolded polypeptide can take on many conformations (large number of possible formations) - BUT, the folded protein can only take on one conformation and is a much more rigid structure - So, the whole protein becomes more ordered when folded, so delta S decreases (more negative)

hydrophobic collapse

- unfolded protein with hydrophobic groups exposed to water collapse so they can be in the core away from water, then secondary and tertiary structures form - part of old view

How does urea denature RNase A in the Anfinsen experiment?

- urea binds preferentially to the denatured protein and disrupts the hydrophobic effect with H bond that are normally important in stabilizing a folded protein

How do you determine sequence of amino residues in tandem MS given the b and y values and mass of aa?

- use only b or only y ions - take the difference in value btwn adjacent peaks on the ms - the difference btwn them = the mass of the aa - use given masses to find which amino acid that mass corresponds to - the largest m/z value (peak- not the difference between peaks) will be closest to the N terminal

How do you calculate the charge on a protein that gives rise to a specific peak in an ESI spec? Given MW and m/z value of peak two away (one btwn them).

- use: m/z = (MW + nH)/n 1. m/z is given, MW is given, H = mass of Hydrogen (1.008, so just 1) and calculate to find n 2. n = charge

describe delta delta G

- value comes from experiments and each aa has one - value is saying how much adding one type of residue into a peptide destabilized the helix - positive values destabilize helix which means less likely to be found in helix (ala, arg, lys) - negative (smaller) values destabilize helix LESS so more likely to be found in helix (Asn, pro, gly)

What is tandem mass spec good for?

- verifying that a protein is what you think it is--> can use it to identify other proteins that may have co-purified with your protein

What opposes folding of protein by contributing to a positive delta H?

- water bonds to polar groups in unfolded protein (H bonds) - some of these groups are on the surface of the protein so folding wouldn't change the bonds broken/ formed (would probably just stay bonded or reform when the protein is folded so overall delta H would be 0) - but, a polar aa residue that ends on the interior of the protein after protein folding would need to bond to another polar aa or a water molecule (less like to H bond to water if on the inside)--> if left unbonded, the breaking but not reforming would contribute to a positive H which opposes folding! - unbound polar or charged residue on interior = decrease in stability and increase in delta H bc not bonded to anything *break bond = + H

Two amino acids you would not insert to avoid changing structure and therefore function and why

1. Glycine: - small and flexible--> can take on many conformations, meaning it can do many phi and psi angles that other residues will not be able to - bc of this, it will most likely change protein structure and therefore function 2. Proline: - rigid and not flexible--> has five member ring that is part of side chain - limited conformations it can take on, especially the phi angle - Pro would likely not fit into same conformations as other residues--> would likely change structure and therefore function

how to determine Kd?

1. mix protein and ligand together **must have less total protein than total ligand present 2. measure the absorbance of [PL] when bonded 3. repeat with total protein concentration the same but vary the Ligand concentration --. measure new [PL] and now have more data points * p total is constant for these experiments - binding curves are hyperbolic (approaches asymptote which approaches total amount of protein in the mixture---> think protein is being saturated with ligand)

Steps to crystalize a protein

1. purify protein 2. take solution of purified protein and add solvents hat make it crystalize - can add buffers, salts, organic solutions, ligands that the protein binds to... 3. pipette protein into wells, add samples, see what above solvent crystalizes protein

what are two characteristics of folding proteins?

1. secondary structures (alpha helix/ beta sheet) form first 2. have hydrophobic core

folding pathways are heirarchical (old way of thinking)

1. secondary structures form first 2. longer range interactions next (hydrophobic effect, involves two secondary structures) 3. complex domains and polypeptide formed

Describe what is happening in the variation of the Anfinsen experiment: Starting: Mix of 100% active RNase A with 4 disulfide bonds - add urea and mercaptoethanol. Describe Mix 2 that results. - remove urea and keep mercap. Describe this Mix 3 that results. - remove mercap Describe Mix 4 that results.

Mix 2: - completely unfolded bc urea disrupts H bonds and mercap disrupts disulfide bonds (completely inactive) Mix 3: - correct tertiary structure bc urea removed (H bonds in polypeptide can form) - disulfide bonds still broken so not completely stable - would be active, bc tertiary structure in tact, but not as stable Mix 4: - folded correctly (H bonds and disulfide bonds) - would be completely active again

describe beta turns

- 4 amino acid residues to make 180 degree turn - H bonds btwn C=O group of residue 1 and NH group of residue 4 --> helps stabilize - residue 2 is usually Pro bc Phi angle around -60 and Pro is rigid structure that can hold the angle (Type I beta turn) - Gly usually at residue 3 bc is is small and flexible and can fit in tight turn (Type II beta turn)

what are the non-covalent interactions that stabilize a Beta sheet?

- H bonds btwn peptide C=O on one strand and H from NH on other strand - Interactions like van der waals btwn the side chains on adjacent Beta strands

number of O2 bonding sites for Hb and Mb

- Hb = 4 O2 bind sites - Mb = 1 O2 bind site

Hb is better for oxygen ___ while Mb is better for oxygen ____

- Hb = transport - Mb = storage

how do detergents cause denaturation?

- detergents like urea bind to surface of proteins and cause them to unfold - the more the protein unfolds, the more surface there is for urea to bind and interferes with non-covalent bonds that stabilize the protein - interferes with hydrophobic effect by displacing water * SDS does similar thing and give protein an overall negative charge

what happens to orientation (stereochemistry) of the R and H groups on the alpha Carbon in a beta sheet?

- each alpha Carbon alternates between pointing up or down - off of the alternating alpha Carbons, the H and R will switch dashes and wedges - so, all R and H groups pointing down could have all dashed R and all wedge H and all R and H groups pointing up will have all wedge R and dashed Hs

How would subbing a larger np residue for a smaller np residue affect Tm?

- larger np hydrophobic residue can make MORE van der waals bonds with the other np residues in interior - more bonds= more stability = increase Tm

m/z peaks that are farthest Right on the spec have a _____ number of charges (n number) than peaks to the left of them.

- less! - so, say peak 1 has n (charge) = 21, the peak to the right of it would have n = 19 and peak to left of it would have n = 20

The more possible starting conformations an unfolded protein has, the ____(greater/ smaller) the entropy change compared to a protein that has less possible starting conformations.

- more starting conformations--> greater the entropy change (will become MORE negative bc becoming more organized); starting more disorganized (high + S) so has to go more negative S to become organized - less starting conformations --> lower the entropy change (will be less negative) bc already starting more organized which equates to a lower entropy

Which FAVORS protein folding? + or - delta H and + or - delta S

- negative H (more bonds forming--> mores stable) - positive S (hydrophobic effect--> np aggregating and hydration shell becoming less organized)

Ka (association constant)

- provides measure of affinity of the ligand for the protein - higher value of ka = increased affinity of ligand to the protein Ka = [PL]/[P] - tells us what is considered "high and low" values for ligand concentration * [L] > Kd--> high [L] and Y is approx 0 * [L] < Kd--> low [L] and Y is low but not 0

ka (rate constant)

- rate constant for association btwn ligand and protein

what are the limitations of a peptide bond?

- restricted to 2 possible conformations (trans and cis) bc of the delocalization of the lone electron pair on the Nitrogen - delocalization (resonance) gives the C-N partial double bond character which restricts rotation

what is the preferred angle of Pro in beta turn?

-60

what are the three types of single bonds in a peptide backbone?

1. N-alpha C 2. alpha C - C=O 3. peptide bond (C=O - NH)

Key points of protein folding

1. Protein function depends on structure (mostly tertiary structure) 2. 3D structure is determined by amino acid sequence (primary structure) 3. Folded proteins: - have one or few stable folded forms - are stabilized by non-covalent interactions - many possible 3D structures but there are common motifs aka beta sheets and helices - conformation changes are important to structure

which mass specs do you use to find molecular wegiht?

ESI and Maldi

Where do charged residues need to be located to form an ionic bond in an alpha helix? in a beta sheet?

alpha helix: - charged residues need to be located three away from each other (1,4,7,10..) to create ionic bond (salt bridge) - should be residue 1 (i) and residue 4 (i+3) - bc helix structure repeats every 3.6 aa residues beta sheet: - would have to be located one residue away (1,3,5,7..) - side chains alternate up or down in a sheet, so want them to be facing the same direction

why is a peptide bond considered both rigid and planar?

rigid: - bc of the delocalization of the lone electron pair on the N of the peptide bond--> creates partial double bond character between N and alpha Carbon which restricts rotation and causes rigidity planar: - bc the electrons involved in resonance are in sp2 hybridized orbitals due to the partial double bond character (pi bond) - to have sp2 hybridization, the bonds the atoms form with have to be in the same plane - all 6 atoms, the C, N, O, H, and 2 alpha C's, are in the same plane - p orbitals need to be lined up to share electron density which means all atoms need to be in the same plane - the sp2 extended conjugated system makes it planar

Size exclusion chromatography separates based on....

size and shape - heavier larger proteins elute first - smaller lighter proteins elute second

when delta delta G is high, the likelihood of it being in an alpha helix (increases or decreases)?

decreases - delta delta G represents how much the presence of that aa in the helix would destabilize it - small number = destabilize it less = more like to be found - large number = destabilize it more = less likely to be found

nucleation condensation model

- the new view of protein folding - both framework/ hydrophobic collapse can happen at the same time, and not all molecules follow the same path

How does tandem MS/MS work?

1. fragment the proteins using a protease like trypsin - trypsin does this on the C terminal side of Lys or Arg (cuts the peptide bond between the C=O and NH by adding water and you get two separate fragments with an H3N and COO ends with the side chains attached) - separated peptides are called tryptic peptides 1a. optional step done with protein mixtures--> separate mix with liquid chromatography 2. MS-1 part: Introduce peptide mixture into MALDI/ESI and separates peptide ions based on m/z 3. select specific prptide ion of interest in frist MS-1--> usually the most present peptide - those enter the collision cell where they collide with heavy inert gas like argon which cause them to randomly break apart - b fragments are the side of the fragment that has from the break point to the N terminus - y fragments are the opposite side of the peptide that has from the break to the C terminus 4. all fragment ions enter the MS-2 which detects m/z from ions and this is the mass you see on the spectrum 5. figure out amino sequence of peptide ion

why shouldn't too many charged residues be back to back in helix?

- bc their charges will repel each other--> destabilize helix

apoprotein

- protein without its prosthetic group - Mb produced without prosthetic group but then gets it

why are Asp and Ser not often found in alpha helices?

- bc they can form H bonds with the peptide backbone--> interfere with the H bonds btwn the side chains that stabilize helix--> BAD

How would the entropy for folding change if RNase was denatured with urea and mercap (than added urea) OR denatured with only urea (then added urea)?

- If the RNase was denatured by both mercap and urea, the denatured protein would be flixble and have many conformations--> why? bc mercap reduces disulfide bonds (strong covalent bonds) and urea disripts H bonds--> together they would cause a larger increase in delta S (more positive) because more disorganized--> when folds, would cause a larger decrease in delta S because going from many possible conformations to a stable conformation that is organized - If RNase was only denatured by urea, the 4 disulfide bond would still be present! this would cause increase stability (covalent strong bonds)--> having the disulfide bonds present would restrict the number of possible conformations the unfolded protein could take on (aka, "less denatured" than one denatured by both things)--> this would cause a smaller change in delta S when folded bc there are less possible conformations to begin with when unfolded bc restrictions by disulfide bonds - so, the change in entropy for the one denatured by both with MORE possible unfolded conformations would be more positive (greater change in entropy from unfolded to folded) than the one denatured with only urea and less possible starting conformations (smaller change in entropy bc already starting off more organized)

Levinthal's paradox

- It is mathematically impossible for protein folding to occur by randomly trying every conformation until the lowest-energy one is found - funnel for this is "golf course"--> idea is that protein can be anywhere on the surface (flat lip of funnel at top) and then falls into the hole (funnel) and reaches the lowest energy conformation (native state)

What happens when a charged residue is on the surface when folded and unfolded? What about buried when folded and unfolded? (ie: Lysine, Histidine, Arginine, Aspartic Acid, Glutamic Acid)

- Lys on surface of folded protein: H bonds to water - Lys on surface of unfolded: H bonds to water *both would yield delta H approx 0 - Lys buried inside folded protein: would have to make a salt bridge to another charged residue--> if not. protein is less stable (positive delta H) - Lys buried on inside of unfolded protein: would make H bonds to water

what two residues are common in Beta turns?

- Pro - Gly

conformations of Hb

- R state: higher O2 affinity in R state - T state: more stable conformation for Hb to be in when oxygen levels are low (dominant form of deoxyhemoglobin) - O2 binds to Hb in T state to one subunit and as O2 binds, the Hb goes from low affinity T state to high affinity R state - allosteric protein (binding at one site affects binding at other sites)

proteostasis

- The continual maintenance of the active set of cellular proteins required under a given set of conditions - includes: folding and refolding - sequestering bad misfolded proteins

How do you tell which protein has higher Tm based on absorbance on a graph?

- Tm is still half the slope - whichever one has higher Tm will have the middle of slope at the higher temp - most likely more absorbance = higher Tm

how would neighboring aromatic residues stabilize each other?

- Van der Waals bonds -(hydrophobic effect)

What specific reactions/ changes occur during protein folding contribute to an overall negative enthalpy (- delta H)?

- a negative H would occur if there are more bonds formed in the folded structure than in the unfolded structure (overall total number of bonds increases) --> remember: bonds forming = decrease in enthalpy! - new bonds formed in the folded protein could include aa side chain interactions, interactions btwn side chains and backbone, Van der Waals, H bonds, ionic bonds * more bonds = more stability = less energy (decrease in enthalpy)

How would a non-polar amino act in folded and unfolded protein- what bonds can it form and how does that contribute to entropy and enthalpy? Does it favor folding? (ie: Valine, Alanine, Isoleucine, Leucine, Methionine, Phenylalanine, Tyrosine, Tryptophan)

- a np residue in the folded state would be buried in the hydrophobic core--> Van der Waals bonds would form btwn the np side chains--> decrease in delta H (bc bonds forming) which favors protein folding - in the unfolded state, the np residues would be surrounded by water molecules, but no bonding would happen bc they are np!--> but, folding would happen bc of the hydrophobic effect which increases entropy of water molecules, which favors protein folding

What specific reactions/ changes occur during protein folding contribute to an overall positive enthalpy (+ delta H)?

- a positive H would occur if there were more bonds in the unfolded protein than in the folded protein (bonds broke when folding and didn't reform) * less bonds = less stability = increase in H/ energy

amyloid fiber

- a protein normally soluble is folded so now insoluble - have aromatic residue concentrations in core of beta sheet or alpha helix

why isn't glycine in helix that often?

- achiral, so flexible so can take on many conformations--> high conformational energy which disfavors folding

in Tandem MS, b ions correspond to what terminal and y ions correspond to what terminal?

- b ions include N terminus - y ions include C terminus

why can only some amino acids be in a beta turn?

- bc a Beta turn is 180 degree turn of polypeptide chain with only 4 residues - sterically cramped! - Pro is favored in the second position on the turn bc of its side chain--> it puts Pro in a good conformation that helps the B turn form; Pro also makes B turn more rigid and stable (favorable here); also favored bc can form a cis-peptide bond - Gly is favored in position 3 bc it is small and flexible and can take on many conformations--> bc B turn so cramped, this is the best residue to fit in the space and complete the turn

why are Beta sheets effective at forming a dimer? (two sheets bonding)

- bc every other peptide bond is pointing towards the outer edge of the sheet--> allows for formation of H bonds between the sheets - the C=O bonds the the H in the NH of the opposite sheet - strands can continue to bind due to H bonding

why can almost any amino acid be found in an alpha helix?

- bc the aa side chains are NOT directly involved with forming the alpha helix - instead, they stick out from the helical core - almost any can do this, but not usually Gly o Pro

Why does delta S decrease in a folded protein?

- bc the folded conformation is more ordered than the unfolded protein conformation

Ramachandran plots for Gly and Pro

Gly: - will have many points bc so flexible, it can take on many conformations - will also have rotational symmetry bc Gly is achiral Pro: - will have few points bc so rigid and has limited number of possible conformations - range for possible phi angles is even more limited - the 5 membered ring causes restriction

True or False: In exchange chromatography, you want the proteins to have large separations of charges.

True--> want fractions to be easily separated while eluting

protease

enzyme that breaks down proteins (catalyzes hydrolysis of peptide bonds)

a rxn for protein-ligand binding with high affinity has: ___Kd, ____ Keq, and ____ delta G a rxn with low affinity has...

high affinity: - Kd is low - Keq is high - delta G is low because Kd is low low affinity: - Kd is high - Keq is low - delta G is high bc Kd is high ** want low Kd for drugs that you want to bind to a protein!

modulators

ligands that change conformation of protein in another subunit when it binds to one subunit - inhibitors or activators - change in conformation occurs is the unstable area of protein

secondary structure

local spatial arrangement of the main chain atoms in a segment of a polypeptide chain (main chain atoms!) - Linus Pauling, Corey and Branson proposed helix