Biochemistry Final Flashcards
9-17
where is glycine a good buffer?
-glycine has two spots where it can go from positive to negative cahrge and experience net charge of zero
- one region where it has a net charge of zero is between about ph 4 adn ph 8, that is where zwitterionic form will be
2 and 9 when moelcules become protonated and deprotonate dthey can act as a buffer they are the moelcules absorbing a hydrogen ion and as a result the ocnentration of hydronium in solution does not change and the pH remains relatively well buffered* around the pKa where these molecules can accept or donate a hydrogen ion to become protonated or deprotonated this is where these molecules will function as a buffer, because glycine has 2 pkas for aminoum and carboxylate 2 ph distint regionds where it will function as a buffer
9-17 Do you think it is energenically favorable or unfavorable to form amide bonds?
so from the 3 aa do you think its exogonic or endergonic? for 3 aa to brign together to form polymer chain energentically unfavorable and will not happen sponateously in fact we find there is a favorable release of free energy when we hydroyzle the amide bond why we develop these tyrosine crystals as we age cheese overtime and individual protein chains begin to degrade into 3 amino acids..?
- Energetically nto favorable to form these amide bodns, take home message: polypeptides are thermodynamically unstable but we have been talking about the fact that thermodynamics is not kinetics, so even though there is a tendency towards hydrolysis of peptide chain but the half life of amide bodn is at ph 7 is 7 yeasr, so kinetically these things are stable and when our cells synthesis protein it is reasonably stable overtime even if present over aqueous conditions when can presumably be hydrolyzed, we will see this over and over again when a molecule or pathway is under kinetic control, that the cell is able to create enzymes that can conditionally speed up these reactions so that the cell is actually able to control the rate at which these reactions can happen
- protein may be kinetically stable, cell may want to degrade particular protein product, cells have mechanism to target proteins for destruction and degrade them at relatively fast rates*
why trans amide bond?
9-17
The answer is that there are steric clashes =
There are steric clashes btw sidechain groups when we have the amide group in the cis confirmation, where we create that zero degrees for the amide bond we will see tht the side chain groups particularly at the c beta position, and create steric clash, that clash is alievaiated if we rotate bond in opposite orientation creating trans amide bond, now we position sidechain groups on opposite side of polypeptide chain, like aleiviating that steric clash just like elevating eclipsing interaction btw methyl and protane??
9-17 why proline 10% cis?
- Polypeptides have a strong preference for this trans arrangement, except for one amino acid in which this preference is not so strong
- Its b/c it’s a cyclic side chain wraps around and forms a covalent bond back to the backbone nitogen
- side chain not only takes off from calpha position it also is attached onto the backbone nitrogen as well, cis-amide in proline is more prevalent than in amides from primary amines
- In the cis orientation this shows the steric clash we tend expect btw two substituents at backbone C alpha positions, but here have to understand that even in the trans confirmation of amide group we still have a steric clash this time its with the portion of proline side chain that is covalently attached to the backbone nitrogen
- So because we have some extent of steric clash in both cis and trans, for proline groups the energeic difference for porlene is less than other 18 amino acids and as a result the energy difference is much less, the prediction would be then if take a look at protein structures, you would expect to see proline residues would have a significant observable population in which the dihedral angle corresponds to cis amide bodn where omega dihedral angle would be zero, that’s exactly what we see, the majority of those amide bonds are trans and omega angle is 180, and we have a significant population of cis amide bonds and protein structures and vast majority of those are formed by proline amino acids* so now we understood omega bond
9-17
phi and psi
- Need to think about types of steric clashes that may come about as we rotate around this phi and psi dihedral angle, if remember amide bond constitutes planes can think of polypeptide chains as a set of these planes connected through hinge elements, we will rotate these planes relative to one another as we rotate these phi and psi dihedral angles
- Rotations in psi and phi determine overall confirmation of backbone, these amide groups can see on plates/planes relatively rigid and we can then rotate these plates relative to one another by rotating around the phi and psi angles
- Again we should be able to identify which bonds are being rotated around as we rotate around phi and psi, rotation of psi is around c-c bond and rotation around phi bond is around the nitrogen c apha position
9-17 Linus Pauiling exam 1 info
- he began to think about how can we put together polypeptide chain that would minimize steric clashes and maximize number of H bonds that can ne formed by the polypeptide amide oxygen NH groups and repeating amide groups in a polypeptide, he was in England caught a cold because of miserable weather lying in bed sickwith his cold began to sketch a polypeptide chain, and tried to fold that piece of paper in such a way to point carbonyl oxygen toward the amide NH group to form an amide bond and tried to see whether he could create a repeating pattern of h bonds in that origami folded piece of paper depening this poly peptide chain, allowed him to predict helical structure of protein
- also i and i+4 positioning!
a. But which dihedral angles are favored in a protein chain? Minimize steric clahses, maximize H bonds!
9-17 Ram plot what he was trying t do
what he did was he began to consider at what angles for psi and phi could he alleviate the steric clashes in a polypeptide chain, and what he saw was that there were actually very specific regions that correspond to low energy structures
9-17
Ram 2
- plotd them out realized oculd make a 2d plot for each position in polypeptide chain that sepcizlies in A psi and A phi dihdeal angles and some of these pairs will alleviate the clashes**
- Plot dark green low energy states, light green more accessible and white denotes high energy confirmations
- Dark green are favorable regions and other regions give rise to clahses and these are disfavored or high energy states, and these plots witht eh dark green show low energy states, light green energetically accessible states and white is the disfavored states, so particular regions in this plot named after him associated with repeating arrangements of the polypeptide chain*
- thser egions, like with blue spots, are part of repeating units that are part of secondary structure elements*
- w e can assocaite them with different secondary strcuture elements, this combination for ex of phi and psi is associate withteh alpha helix* etc.
9-17 alpha helix
-Repeating pair of phi and psi dihedral angles do it at every position create a helix, see one full turn of polypeptide chain every 3.6 residues within the protein
-We also see that as we create this particular arranagment we position backbone atoms to form a repeating network of h bonds btw amide carbonyl oxygens and amide h groups oriented in exact arrangement that can give rise to this network of hydrogen bonds
What defines an alpha helix=phi and psi dihedral angles about negative 60-50 in Rha’s plot allowed regions, we have all amide H and carboxy groups hydrogen bodned to one another except for those at the end of the helix for which there is no particular partner, as a result the termini of alpha helixes have exposed amide /NH and exposed carbonyl oxygens and these tend to be POLAR and located on the surface of proteins8 tned ot be locate don the surfaces of proteins
e. so our characteristic pattern of hydrogen bonds if we take the carbonyl oxygen of one amide group and create a hydrogen bond in this arrangement specified by the alpha helix we will make the h bond with the nh group of the residue at the 4th position down the chain, take that carbonyl walk 1,2,3,4 down chain form h bond to that Nh group creates regular pattern, do that at each and every position as go down the peptide chain, we make that network repeating hydrogen bonds and that is what defines an alpha helix, see this all the time I to i+4
9-17 left handed helix
- we see this rarely becuase it is such high energy state
9-17
i to i+3/ or i+4
- i is at close proximity of side chains as positions i+3 to i+4 amide groups in backboen but also possibility to create interactions between chemical groups within th die chains, which amino acids at position i are going to interact with i+3 and i+4 becuase they are on teh same side of that cylinder formed by the helix SOO side chain groups can then pariticipate in electrostatic interactions, pie stacking and van der waals intearctions*
Side chains at i position can interact with side chains at i+3/i+4 positions using salt-bridges, hydrogen bonding, pi-stacking or van der Waals interactions
9-17 alpha helix hydrophobic/hydrophilic details
- Helices are often amphipathic
- Helices often have a hydrophobic face, which points into the protein interior, and more charged/polar faces, which point into solution.
- this turns out to be very nice when packing secondary elements into protein structure, in this situation take individual helix and barring it into the interior of folded proteins, we see a typical arrangement will be for all of the amino acids to be hydrophobic very foten we will see helix at the surface where one face of the helix is exposed to solvent other face of helix is pointing inward towards protein so tends to be amphiphilic
- orient amide groups along alpha helix position to create H bonds to one another as we do that we align individual dipoles in each amide group and get them all pointing in the same direction, as a result overall dipole associated with alpha helix call that a helix dipole
9-17
helical wheel
- the charged and polar groups oritented on one side of the helical wheel and other side is hydrophobic residues other individual helices fully exposed to solvent very often all of those amino acids around the helix are fully charged and polar amino acids
- 3.6 residues per turn
- Two turns of the helix bring sidechains into the same projection from the helix axis
- Residues at i, to i + 7 are in proximity
9-17
Linus pauling i and i+4
- he though about how he can fold up this chain so that every oxygen is associated with a H bond with soem other NH group in the chain, realized forvable arrangement but also instead of bending paper aroudn could also bend it around to go from i+3 amide NH group results in different secondary structure will talk about in next lecture
9-17
Some residues prefer to be in the helical conformation more than others
- Helix propensity is favored by Ala, destabilized by Gly or Pro.
- Leu stabilizes helix more than I or V. Crowding Cβ is less favorable.
- Side chains interact at spacing of i,i+3 and i,i+4: acid base bridges (E,D to K,R) stabilize helix, hydrophobic groups too.
rectiation 2pm 9-28
know diff btw chaperonins and chaperons, what are their mechanisms of action, what stage they act
- know for CHAPEroNES* which are typically smaller proteins they actually do not REFOLD misfolded proteins, hwo they act during the process of protein folding, the chaperones bind to specific some patches of aa along backbone of polypeptide, they help prevent misfolding of proteins so chaperones do not refold misfolded proteins they help a polypeptide chain fold to its correct confirmation by preventing misfolded states from being populated, the ATP hydrolysis in this case is used for a slightly different purpose, chaperones actually use energy of ATP hydrolsys to undergo confirmational change to either bind to polypeptide or release polypeptde
- but with chaperinons each 7 subunits within the chamber hydrolzyign atp to undergo also confirmational change but in that case each confirmational change created a new interaction with polypeptide chain so chain either extending out or getting a chance to refold back**
9-15 I think?
addition of salt for ex can be used to alter protein structure and participate proteins as we purify them?
Yes because salts have positive and negative charges, if you out a bunch of negative charges by the protein cause poisitive chrges t be attract t them or opposite so that can change the confirmation of the protein that way
Groel vs GRoES-
confirmation change that chamber undergoes, as ATP hydroylzed to ADP chamber expands and as it expands expsoes hydrophobic surface area on the interior, that is complementary to the hdyriphobic groups in the improperly folded protein now gives that improeply folded proteins ubstrate and opportunity to open up to detach hydrophobic interactions that are intermolcualr and incorrect and reasocate them with the envirornment* this in essence unfolds the protein from a properly folded sate, once it does that essentially whats going on it has been puled back up to top of that energy funnel** folding funnel he showed us before** so that hydrolysis of atp is paying energy to create a new environment inside that chamber and to pull that protein into an expanded form from which it can try again to fold properly*** partial polypeptide chain pulled apart, and let fo pulled apart and let fgo- while that happens in lower chamber ti chamber left open, not happening in top
champeron versus champeronin
- Any small protein, dimer- clamp like chameprone
- Any big like structure whith chamber like structure inside it is champeronin
- Clamplike chaperones
heat shock proteins
- Small proteins, ehat shock proteins because way discovered when ppl raised the growth temerpature of bacteria cultures to higher temperatures saw expression of proteins in bacterial cells, as if bacterial cells trying to do something to mitigate effect of inc temperature, so we know when temp of system high proteins start to be unstable or amy unfolded protein has harder time folding into its structure
- So these proteins called heat shock proteins were making higher amounts inside bacterial cells to prevent misfolding of other proteins or to help other proteins folding into the correct structure** so first tthign to remember HSP heat shock protein DO NTO REFOLD MIS FODLED PROTEINS*****
- They act DURING the folding reaction of a polyp chain to prevent chainf rom going to misfolded state, or they try to bias folding reaction ofpolypepyide chain toward folded structure, if piolyup misfolded cannto rescule polypepyides into correctly folded structure
aggregate proteins why need chaperons
- So say a polypeptide is folding into its 3 structure, generates intermediate structure say a bunch residues unstructured still say many are hydrophobic regions, these hydrophobic regions instead of forming hydrophobic interior cluster, hydrophobic regions btw diff polyp chains can nonspeiically interact with each other to give rise to a bunch of aggregates proteins, thse aggregates are not stabilized by intarmolecualr interactiosn they are stabilized by INTER moelcuarl lnteractions btw two polypeptide chains by hydrophobic residues by some other interaction generating aggregates
- Polypeptides in misfolded state in aggregates have no use to us, in order to prevent the folding intermediates or actual initial protein starting protein reaction to form aggregates what we can do if have certain prptein grabs onto folding polypeptide chain covers up region that can potentially undergo intermolecular interactiosn what will happen is these clamp proteins prevent intermeolcaurl interactiosn from happening and gives polypepide chance to generate intramoelcualr itneractiosn for correct strucurre or drive protein to right pathway by forming right pathway itnermediates, grab on undergo confirmational change, clamp like champerones
chaperones
are in more extended confirmation when bond to atp, when interact in open bdingin cliff interact with bidnign peptides can either bidn to completely unfolded peptides or trapped folded intermeidiates
-once interact with bidnign clamp, hydrolysis ATP undergoes and results in confirmational change in clamp like proteins that go from closed state to an open state, in that closed state it has a much higher affinity for plypeptide bidnign to, ithodsl onto the polypeptide, rest gets a chance ot generate folded structure or intetermeidate, and after a little bit a new protein come sin calld ncueltoide change factor kicks out ADP generated by hydrolysis of atp chamber like protein and replaces it with another atp molecule then clamp like goes back ot original confirmation elases paritally foled or almost totally foldd protein, then protein biased to form intermolecular contacts to give rise to native structure and clamp like champerone is recycled boudn to atp and in open cofniramtion when this another poilypeptide can hit another protein and go through same process
o Pulloing entire reaction towards favorign folded state of protein because its preventing surfaced exposed regins of folding intermediates ot fold itnermeocluarl interactions that can lead ot aggregations ormisfodld structures of intermediates*
o If already misfolded calmp cant do anything, but they can bind to it and deliver to chaperoneins
Two conditions happen if misfolded:
If conditions of cell are not condusive to recovering misfolded protein then prteins can be sent to degregation pathway were certain small tags given to protein sent to proteosome death chamber, under certain conditions instead of degrading proteins given a second chance to correctly fold back into native structure
o A bunch of chaperones bidn to these misofodled structure and send them to chaperonins*
How do you ensure only misfolded protein gets into GorEL complex??
So what happens is when proteins misfolding happens which is a huge area of study by itself, when proteins are NOT in their most stable confirmation in their cell, they have certain residues* that are exposed on tehri surface, in many cases many different types of residues exposed many cases hydrophobic residues typically should be buried inside protein, because of misfolded or stable misfolded intermediate they are exposed on surface, so there are certain proteins which just like the chaperones we talked about before this, groel groes examples of chaperonin proteins, also something called chaperons that help proteins fold, smaller soluble proteins that help other proteins fold o class of chaperoines that bind to hydrophobic surface of misfolded proteins and delivery these misfolded proteins to these chambers in the absence of these additional chaperon proteins, the chances of the misfolded protein being delivered to the groel groes complex would be much lower as opposed to higher probability of being degraded, if proteins chaperons not to bind and deliver to champeronins those misfoled proteins would actually be sent to different protein chamber inside the cell called proteosome where they are degraded, death chamber o there is a continuous partitioning of misfolded proteins between either going to degraded pathway or chaperon mediated refolding pathways**