Protein Biochemistry Midterm I
Then does same experiment but instead of going from water to ethanol what if we go from water to urea. Urea downplays any hydrogen binding interaction so H effect is less significant. So if we use urea the DeltaG transfer should be less negative and less favorable... basically saying the hydrophobic effect is less significant in urea
8M urea is a slightly better solvent for hydrophobic molecules than pure water
Creates a force where any chemical system will limit the amount of interface it is - bc it is energetically unfavorable (reason for water droplets/oil droplets) Why hydrophobic droplets want to come together because they will decrease the amount of water cages needed
A potential increase in entropy represents a strong driving force: the hydrophobic effect
Alzheimer's disease is characterized by the formation of insoluble plaques composed of amyloid protein A𝛃 forms packed 𝛃-sheet structures Alzheimer amyloid is formed by aggregates of an ~40 amino-acid peptide (A𝛃) derived from the APP protein
Alzheimer's disease
Amino acids in proteins are linked through peptide bonds - Amide bond (condensation reaction) - Amino acid addition occurs at carboxy terminus of a growing peptide
Amino acids in proteins are linked through
- Amine (NH2) and carboxylic acid (COOH) - Ionized at neutral pH (zwitterion) - Cα is chiral in most cases (L amino acids; except glycine) - Side chain (R) defines specific character - Proteins are produced as linear polymers of amino acids - Genetic code codes for 20 different side chains
Amino acids: basic building blocks of proteins
Amphipathic helices are a major building block of transmembrane channels hydrophilic face forms channel for ions or water molecules hydrophobic face interacts with the fatty acids of the membrane
Amphipathic helices
Requirements: 1)Unique free energy minimum. 2)Stable - free energy around minimum must be steep and high 3)Kinetically accessible - energy surface not too rough (no large activation energy humps) Everything needed for protein to fold is contained within primary structure. Can only be amino acid sequence itself* Not all proteins follow anfinsen's dogma bc not all proteins have global energy minimum Unique: one energy minimum Stable: cant have several possible confirmations at one minumum Kinetically accessible: no large activation energy humps Chemically synthesized peptides fold similarly to biological peptides, confirming that amino acid sequence decides structure.**
Anfinsen's Dogma
Both conformations have essentially complete backbone H-bonding can do H binding nicely bc pretty much in straight line Anti-parallel stacks more nicely and lines up in comparison to parallel (can see the bond is more at an angle rather than across)
Anti-parallel β-strands and parallel β-strands
- Very little energy is necessary to transition between related states - Small changes: small side chain rotations, backbone 'breathing' Doesn't mean its in one state at this point: will still jiggle but with small eenrgy changes (little confirmatons within native state - side chains rotating for example) We think of idea that there is one perfect folded energy confirmation but there is several different variations and will fluxuate its structure just a little bit. Small rotations and not just folded there is still a little bit of rotation
Any protein at room temperature exists in an ensemble of related but different states
As concentration of denaturant (or temperature) increases, the chance of unfolding increases Every point in this graph represents a dynamic equilibrium. Individual proteins can transition between folded and unfolded states. However, the total fraction of proteins in the folded and unfolded states stays constant Denaturant can also be increasing temperature. There will point where protein is reduced enough that it is no longer favorable to be folded so it becomes unfolded. The half point is where half protein is folded/unfolded, it is where equilibrium is at. Folding is a super fast process so measuring a dynamic population but overall proportions will stay the same
As concentration of denaturant (or temperature) increases....
The concentrations of reactants and products no longer change. Important: forward and reverse reactions still occur, but at equal rates!
At equilibrium the ratio of reactants and products remains constant
Interactions of hydrophobic side chains with charged or polar molecules (e.g. water) are energetically unfavorable (hyrophobic effect). Close packing of hydrophobic side chains contributes to protein stability. Can interact with each other, but not with water Enriched on inside of proteins: hold proteins together and keep them stable
Big and hydrophobic amino acids are enriched in the protein core
Favorable in this case to unfold more. But folding can still occur*** at any point which will drive reaction toward eqilibirum You will be unfolding but saying forward rate is stronger than back rate you will still have protein folding The folding reaction can occur at some level even if it is energetically unfavorable. However, overall the unfolding reaction predominates as long as the ∆G for the F→U reaction is negative. This imbalance drives the reaction towards equilibrium.
Can folding occur in this situation?
Within molecule much tighter binding so on bottom the positive charges within histidine competes and wins (binding) over the yellow other molecule
Changes in charge state of histidines control the binding and release of LDL from its receptor
(e.g. 8M urea, 6M guanidinium chloride) Increase the solubility of non-polar substances in water Urea: makes H bonds less of a benefit and as a result water doesn't matter so less reason to make them come together. Distubrs H bonding and disrupts hydrophobic affect
Chaotropic solutes in water
-Low sequence complexity -High proportion of polar and charged amino acids -Low proportion of bulky hydrophobic amino acids
Characteristics of natively disordered regions
Charged amino acids can form stable electrostatic interactions between side chains Although electrostatic interactions can form at any angle, the interaction between positively and negatively charged amino acids also involves H-bonds. The combination of electrostatic interactions and H-bonds is known as "salt bridge"
Charged amino acids can form....
Circular dichroism measures the differential absorbance of circularly polarized light This is iportant for optically active complexes (all amino acids but gly) so will inteact with light. Secondary strucutres also react extremely strongly. Somehow you can put something on light wave and so it rotates in clockwise fashion = Cricularly polarized light. You can polarize light in both directions (clockwise and ccounterclockwise) and if im a molecule there is a difference in how I would interact (absrob) clockwise vs counterclockwise light. The difference between the two is ellipticity.
Circular dichroism
Collagen is made up of three polyproline II helices coiling around each other Most abundant protein in mammals High tensile strength (Gly-Pro-hydroxyPro) repeat Glycines pack in the center of a triple coiled coil Proline helixes = how collagen is made Hyp = modified proline, helps keep collagen stable
Collagen
Important: The pKa is a constant. This means if pH (the concentration of H+ ions) changes, the relative concentrations of X− and XH must change to compensate. Thus: Lowering the pH of the solution (i.e. providing more H+ ions) will force more X− into the protonated XH form. Conversely, increasing the pH will drive dissociation of H+ ions from XH.
Conjugate acid-base pairs exist in an equilibrium described by the pKa
Proximity helps search for binding partner - proximity will affect how something will fold. Because of finding things at different times: they can be stuck at certain points (kinetically trapped) if they found a good partner but still not at optimal Part of protein will fold locally and ideal native state and then other parts of the protein will fold. Whole long thing we see that these amino acids fold into each helix. All amino acids will form a local secondary structure then tertiary starts to develop Most hydrogen bonds form between residues close in primary sequence -near-neighbor backbone interactions (e.g. α-helices, loops, antiparallel β-strands) -side-chain H-bonds similarly form more commonly with close neighbors
Contact order is correlated with folding rate
polymer (4 types of monomers) double-helix formation by base pairing several types of non-B-form DNA stable and rigid backbone (The potential to encode structural information differs strongly between biomolecules)
DNA
- proteins unfolded and relatively uniformly covered with charged detergent - separates primarily based on mass - used to determine protein size
Denaturing (SDS) PAGE
disrupt proteins of bacteria, disrupts hydrophobic effect and exposes them which kills them one polar and one nonpolar side.
Detergents:
Waiting for equilibirum in each case. We get different eqilibrium constants for each. Changing Temp and Keq and deltaG is constant Temp changes rate constant** EXAM (x-axis: temperature — this is an equilibrium experiment; we wait until reaction is done and then measure)
Determining the free energy of (un)folding
Even though we don't do direct measurement with etahnol/water we can calculate the difference and calculate the ∆Gtransfer which tells us the energy from putting leu in different environment If you want to look at side chain: COMPARPE against glycine** (which energy is from amino acid itself vs its side chain) Full circle should be deltaG of zero. We wanna see methane group (ala or leu) and go to ethanol and see how much energy is released but this is physically impossible. What you could do is take leucine as a powder and put in water and see free energy released or absorbed and separtely drop Leu powder in ethanol and see free energy and then you can deduce that the ∆Gtransfer would be the difference between the two. WE CAN do see the extent hydropbic effect for individual amino aids******* We cant measure it directly but this is how we can
Determining the transfer energy from water into ethanol
Dialysis allows the selective dilution or elimination of small molecules Semi-permeable membrane lets small solutes pass but is not permeable to large molecules. Youre exchaning the buffer and basically got rid of all urea and B-mercaptoethanol If you have semipermeable contained and you have bunch of salt eventually it will leak out if you have big enough container. You can continually put into new bags of water. If you put into fresh bag because of entropy they are likely to leave. Ex: isolated protein and expose to urea and bmercaptoehtanol and then expose to water all of the urea can leave through membreane but protein cant bc too big and then urea and B-mercap leaves and now we can see what happens
Dialysis allows for buffer exchange
The disulfide bonds stabilize the structure and maintain the connection between the two peptides after proteolytic cleavage of proinsulin.
Disulfide bonds allow amino acid sequences to be stably (covalently) linked to each other
All linker needs to do is coil in some way - only thing cant do is get too hydrophobic. Now lots of freedom in term of evolution - mutations will not affect it in the linker, but if in other parts of the protein - there would be a decrease in stability. Domain is super folded so you have linkers between each domain
Domain boundaries can often be assigned based on sequence conservation
-Allosteric effectors do not directly bind to active site -They use domain flexibility to alter active site structure at a distance Active site in a protein but you can regulate how easy this active site is availble by allowing another thing to come in - can lock in inactive or active conformation = allosteric regulation
Domain interface flexibility also provides a way for "allosteric" regulation of enzymes
Red = backbone, small hydrophobic core (not enough to hold it together), but zinc is making complex bonds 10 amino acids for example enough: about 40 is a good value
Domain sizes are dictated by folding energetics
-on average ~100 residues (~11 kDa) in size -independently fold into stable tertiary structures -often independent functional units -same domain may be found in different proteins -evolutionary building blocks of proteins -indenpendelty folds -own function -can cut and paste them
Domains
Connect whatever receptor you need and different domains that signal this. You can modify these domains without affecting the catalysis itself*** just what it interacts with Tiny changes in amino acids can have similar function but will change what it interacts with
Domains are reused in different proteins by evolutionary mixing and matching
Domains often have different functions Domains to give it structure and help it dimerize Evolutionarily if you want to make a new binding domain you can just switch out the DNA binding domain and keep the others This is much more common in eukaryotes
Domains can usually be associated with specific functions
What youre looking for is that there is some change/folding. We not increase the denaturant - IF INDEPENDENTLY FOLDING: it will be stable stable stable until you hit deltaG then will increase** COOPERATIVE AFFECT, domains fold apart (cooperative transition). If not independent, it will be not folded already and wouldn't be a change in CD or florescent - there would be no cooperative transition. Key is S shape. To measure protein folding: CD and florescence quenching To see how many domains? We can could number of transitions so we know its two domains (right). We cant be sure that blue is just one domain: they could have the same temp at which it unfolds so cant tell if its more than one. This tells us the minimum of each domain
Domains display characteristic folding behavior
Domains are much narrower bc demands on folding. Need to be stable but also occur quickly Median size: domain is 50 and protein is a little over 200
Domains fall into a relatively narrow size distribution
Domains provide a way to break down the folding problem for proteins
Domains provide....
N-terminal protein sequences of fragments can be determined by Edman degradation PIPTC (n-terminal) does nucleophilic attack and form covalent bond and now another nucleophilic attack on carbonyl group and you hydrolyzed what amino acid you want. Then you analyze the the ring (left) and then analyze the amino acid
Edman degradation
Edman degradation requires a free amino group*** Why we don't do this as much as we could? /useful but not used a lot -you need lot of material -most proteins don't have free amines, most amines are acetylated -side reaction with PITC (lysine can also react with this so can get in the way)
Edman degradation requires....
Several amino acids, protein modifications, and elements exist in an ionized state at biologically relevant pH levels. know + vs - interactions
Electrostatic interactions
Net extent of force is decreased in highly polar solvents (𝝐water = 80, 𝝐hexane = 2). Strength of electric interaction depends on charges, and dialectic constant (E)
Energy of electrostatic interactions
There isnt really a global energy minimum bc all of these states are stable. Theres a broad valley instead of a sharp minimum so don't adhere to ***dogma
Entire proteins can be intrinsically disordered
Entropy measures the degrees of freedom (the "disorder") of a system Smaller number of possible H-bonding interactions = less freedom = decreased entropy = energetically unfavorable Nonpolar nonpolar bidning is stronger than charges/ even polar-polar binding
Entropy
Equilibrium ratio of protonated and unprotonated forms of a compound depends on the pH -The ionization constant pKa describes a compound's affinity for protons (H+). -The pKa is the pH at which 50% of XH is dissociated into X− and H+. -Each conjugate acid-base pair has a characteristic pKa.
Equilibrium ratio of protonated and unprotonated forms of a compound depends on..
1) Highly conserved (ancient) roles in the cell - tRNAs - Ribosome - Signal-recognition particle 2) RNA catalysis during protein synthesis and RNA modification - Ribosome - Self-splicing introns - Other ribozymes 3) RNA-related molecules are widely used co-factors - ATP, GTP - NAD+, FAD - Coenzyme A Highly conserved steps done by RNA**
Evidence for an ancient RNA world
From partislly folded state to fully folded you need to put in energy again. Need to give enough energy to get over hump so can continue with the folding pattern Here: overall reaction is favorable Activation energy height: tells us how fast something is going to happen - if small = high chance of getting F.
Exit from a local energy minimum requires activation energy to cross the energy barrier
Ribozymes can be made that use a template to synthesize RNA equal to their own length (reaction takes several days).
Experimental evidence indicates that an RNA world may be possible
Kinetics experiment Theres some that fold fast but only 75% are folded Lots of different variations: some biochemist job is to make more stable versions. Some don't fully fold to their native state: nto that taking a while to get to state its that its plateus and is representative of a metastable confirmation so must be stuck in another semifolded state
Experimental evolution yielded faster folding forms but some experienced kinetic trapping
Fluorescence quenching Circular dichroism (CD) UV-Raman resonance Nuclear magnetic resonance (NMR)
Experimental techniques to study protein folding kinetics
Has a bunch of disulfide bonds (normal fold is stabilzed by sulfur covalent bonds). For this to unfold we need urea AND reducing agent to break disulfide bonds to now be unfolded. You can let it refold by removing the urea and B-mercapatoethanol. Urea: makes H bonds worthless so helps denature so breaks these bonds - super denatured
Experimental unfolding of ribonuclease A
1. Absorption/excitation 2. Excited state lifetime 3. Emission High wavelength = lower energy Stokes shift = different in wavelength which tells us difference in energy
Fluorescence
Fluorescence of tryptophan depends on the hydrophobicity of the environment As long as they have tyrosine and tryptophan we can use this to see protein without unfolding them. If trp is in hydropobic region it will give off light with higher energy than in water Bascially using this as a probe and depends on what medium it surrounds If you hit photon with a light beam and its on inside it will send out a bunch of light and reamit photon very well. If on outside of protein it does a worst job of sending photon back. Trp is hydrophobic typically so normally on inside
Fluorescence of tryptophan depends on.....
Fluorescence quenching: when trp and tyr are in hydrophobic environment it floresces well and will go from pretty good florence and then it will unfold and there will be lower floresence
Fluorescence quenching
whipped egg white example, now air pockets that hydrophobic residues stick into Foaming: bringing in something nonpolar and hydrophobic effect is destroyed
Foaming
Fold: Super-secondary structure found in a variety of proteins Domain:Experimental: an independently folding section of a protein Computational: a distinct protein sequence conserved in evolution Difference between fold and domain is that DOMAMIN FOLDS BY ITSELF Bc domain are functional units there are less mutations there bc they cant change too much so there is less freedom to evolve. Conservvation can allow us to see evolution. Zinc finger os an example: reason it folds is bc of zinc ion Motif/fold: super secondary structures is more of a common interaction of secondary structures, if the same way alpha helices is a common interaction. If we continue logic how many different ways can they interact? Helix loop helix. Telling us that something is a helix-loop-helix doesn't tell us anything about its function just that it's a patter Domain: functional and folding significance
Fold versus domain
Folding can be represented as a funnel because ∆G is independent of the reaction path Note some paths may be practically nonexistent: - kinetic (high energy barrier) - structural (e.g. backbones cannot pass through each other)
Folding can be represented as a funnel because....
For unimolecular reactions, the reaction constant k is equivalent to a frequency
For unimolecular reactions
know strength*
Forces that stabilize protein structure and binding interactions
Proteins migrate in an electric field through a defined gel meshwork; separates proteins based on charge and bulk.
Gel electrophoresis of proteins
Glycine and proline occupy extremes in the energy landscape - Absence of a side chain in glycine allows more angles - The ring structure of proline strongly restricts favorable angles - Proline is incompatible with α-helical conformation ("helix breaker") Glycine: more space to bind, no side chain so can explore much more space due to less steric hinderance Proline: N in ring so cant explore space anymore, very limited possibilities = helix breaker
Glycine and proline
H-bonds are strongest when approximately straight Chemical systems always favor the lowest-energy states. Energy for hydrogen bond formation increases steeply for non-linear angles of NH..O geometry
H-bonds are strongest when..
helical wheel representation helps visualize the orientation of side chains Helical wheel" representation: Looking down the helix from the top **Every 7th residue is at approximately the same angle from the helical axis.**Usually repeats itself after every 7th
Helical wheel" representation:
Experimental: an independently folding section of a protein Computational: a distinct protein sequence conserved in evolution
How do we know a particular protein fragment is a domain?
Hydrogen bonds are the only non-covalent interactions that have directionality Anything that has H and a negative charge = acceptors Free electron pairs = donors - H-bonds have electrostatic as well as covalent characteristics - H...O=C angle can be between 120° and 180° - Energies of H-bonds range from 4-40 kJ mol-1
Hydrogen bonds are the only non-covalent interactions that.....
Held together all by hydrophobic interactions Leucine zipper is a coiled coil: just when alpha helices wrap around each other 1 and 4 could be nonpolar so would wrap up bc hydrophobic
Hydrophobic amino acids form the helical interface of coiled coils and leucine zipper motifs
Hydrophobic effect: water molecules have to "stand guard" - bad bc cant move. Unhappy bc of entropy. If there is two so there would be two unhappy water molecules. So instead, these fat molecules would move bc would reduce surface area and help water be a little more happy and increasing entropy. ***freeing water molecules so they can have rotational freedom which is good for entropy
Hydrophobic effect
Increased order of water at hydrophobic interfaces is energetically unfavorable. entropic effect
Hydrophobic molecules limit possible hydrogen bonding interactions for surrounding water
How to sequence amino aicds** Commonly use Mass spectrometry: works bc we are weighing the amino acids. However we need to know sequence before starting.
Identification of proteolytic fragments
Metamorphic proteins can exist in two stable structures of equivalent energy Metamorphic: can exist as two stable structures and both minimums are relatively comparable - there is an equilibrium between these two There might be two equally stable folded onfirmations: not one true best folded there's two best states - how one gets to other can depend on pH or other proteins
In rare cases proteins can have two equivalent global energy minima
Proteins may fold into their native structure under dilute in vitro conditions but may not be able to do so under crowded conditions. Proteins don't just come out of ribosomes at once: come out gradually (what the dogma didn't consider). In terms of folding it's a different folding path: all depends on what comes out of the ribosome first When proteins first come out: -hydrophobic parts stick together - dangerous Main function of chaperone: prevent them from sticking together**
In the crowded environment of the cell, nascent and unfolded proteins are at risk for aggregation
Virus is taken up by endocytosis. The lower pH of endosomes creates a now global energy minimum that changes the structure of hemagglutinin. If cell takes virus up: the virus wants to get out of endocytosis membrane and has membrane penetrating domain to it which empties out stuff into the cytoplasm but is a pH SENSOR: waits until it gets taken up and now it's a new global energy minimum and will intiate the punch holes Protein that has two different native folds, the difference of why there is one vs another the pH changes its favored folded state: virus comes and binds to surface protein and can trick cell for endocytosis. Changes confirmation once endocytosied based on pH changes
Influenza hemagglutinin assumes two different native folds depending on the pH
Initial collapse is driven by hydrophobic effect and formation of alpha helices Optimization of hydrogen bonding and van der Waals interactions Biggest release of energy is the intitial and then molten to stable takes a little longer Unfolded protein in bad pH and mix it with correct solvent and now it starts to fold. Can see whats folding and how: combining both. First thing to cause the fold/collpase is hydrophobic effect and formation of alpha helices then you have others
Initial hydrophobic collapse and helix formation can follow many related trajectories
pKa = −log([X−][H+]/[XH])
Ionization constant:
Isoelectric focusing uses the pI to separate proteins in an electric field Proteins will migrate until they reach a position in the gel strip that matches their isoelectric point
Isoelectric focusing
Measure reactions as a function of time Always need: - identities of participants and products - proposed reaction mechanism A few molecules once folded have a chance of unfolding Say we have 100 molecules of A that are turning into B. After a few seconds the rate falls and falls
Kinetic experiments
D- amino acids not used: bacteria use for cell wall
L vs D amino acid
Pure protein and stained with dye. 2: protein without treatment THEN add proteases and then stop reaction at different times and see the breakdown products - now we have two band: two different domains. If we wait even longer, we see new bands but not really domains it'll just be other cuts but not distinguish domains. Trypsin: good for basic emotion acids Chymotrypsin: amino acids YWF are hydrohobic so not in linker so would be much harder to cut We don't have order so we don't really know where the domain boundary is - but we can get at amino acid sequence of these two structures. CHOPPING UP RELEASES FREE AMINE. So if amine is acetylated this experiement will free up one amino acid then you can do trick from next side to see identity of amino acid**** Protein with w/ two domains and what we have is that it is cut into two chunks and can tell us probably how many domains.
Limited proteolysis can help define domain boundaries
Proteases: cut peptide backbone. Linkers often solvate nicely and are readily accesible byother enzymes. If you add proteases they will cut at the linker (much harder to cut at the backbone) After proteases the units are all separated and see where the boudnaries of the domains are. Proteolysis will happen here. -Proteases require access to the peptide backbone for proteolysis -Unstructured linker peptides (loops) are most vulnerable to proteolytic cleavage -Allows mapping of domain boundaries
Limited proteolysis can help to experimentally define domain boundaries
vesicle and bilayer formation membrane curvature, thickness and fluidity non-polymeric (The potential to encode structural information differs strongly between biomolecules)
Lipids
This extends the reach of the protein - sometimes hard to find binding partner so can help explore more space. Appropriate confirmation is caught later. If binds interacting partner it can real it in and form its structure. This can help multiple interactions with same domain Whole proteins can do the same: we cant determine structures so we can digure out whats the smallest/largest thing that can form: different types of confirmationt hat is consistent with what is known Rg = radius of giration Allows them to find eachother and pull them together The appropriate conformation is selected through interaction with an interaction partner (conformational selection) Disorder is also thought to permit interactions with multiple interaction partners.
Long unstructured tails are thought to increase the chance of binding an interaction partner
β-ME acts as a catalyst: It facilitates a reaction but is recovered unchanged at the end of the reaction. Regenerates B-ME which is why it's a catalyst. All this is is trying to get to lowest energy (it was trapped in not its lowest)
Low levels of β-ME unlock disulfide bonds and allow RNase A to reach its native conformation
Any single bond can rotate. Relative positions are strongly dictated by spatial (steric) constraints. Amide bond pretty much never rotates
Many bonds in the peptide chain can rotate
Disordered regions are a very common feature of proteins involved in interactions with RNA or DNA, and in signaling complexes. pKID domain: not really domain bc not independently folded only folds in presence of cofactor. Does have its own structure but doesn't do it on its own
Many disordered regions only assume a stable structure in conjunction with a binding partner
We don't know order - fragmentation will break at peptide bonds and you get -E for example and then you can deduce that E is first. Different peaks = chopped up parts of peptide so you can read off bits off sequence y axis is abundance so at 1400(whole length) youll never have bigger than 1400 Most abundant = most stable product when we hvd this level of ionization break it apart
Mass spectrometry can be used to determine peptide sequences
Problem: Dissolution in ethanol is a better approximation of the hydrophobic core, but EtOH and water are miscible - a direct transfer reaction is not possible What is hydrophobic effect for one particular amino acid? What you could do is take a methane to represent an alanine and take from water to carbontetracholoride (moving environments) and see how much energy is released or absorbed Would be better if we did in ethanol but cant mix this so cant do this directly:
Measuring the ∆G of solubilizing hydrophobic side chains in water
Channel is perfect positioned to mimick oxygens around K, so K can let go of water and stabilized by channel = NO ENERGETIC COST If Na, not in right position and would cost energy Hydration shell for potassium (K) is perfectly mimicked by channel Backbone carbonyls in the pore are precisely placed to match the distance of the oxygens in the hydration shell of K+. → essentially no change in energy. By contrast, the distance in the channel mimics partial desolvation of Na+. → energetic cost to leave water shell.
Mechanism of ion selectivity and transport in resting K+ channels
Molecular chaperones use the energy of ATP hydrolysis to capture exposed hydrophobic peptides and assist in protein folding. Typically binds to hydrophobic segment and typically requires ATP: helping it fold quickly without damaging other things.
Molecular chaperones protect native and unfolded proteins from aggregation and promote folding
Bacterial cells: have these things close together different genes into one 5 different proteins that specifically interact with same substrate to cause specific response: Eukaryotes just make one giant protein that had 5 domain so we don't have to get everything together
Multi-domain organization is a common feature of eukaryotic proteins
Tm goes down as ∆∆Gº goes up = destabilizing
Mutations affecting ∆G° will shift the melting temperature (temperature of unfolding)
Trp = steric interferance and value goes up even more Pro = breaks alpha helices so will go up a lot on exam prob
Mutations of single amino acids can severely reduce protein stability
NMR doesn't depend on crystals: take advantage of the fact of how close nucleus are together: dependent on the magnetic field. Will react with each other and will give you a hint on the spacing of individual atoms PROBLEM w NMR: there's so many atoms so spectra comes back super messy so hard to analyze.
NMR also provides information on ensembles of disordered structures
- to separate folded (=native) proteins and protein complexes - depends on isoelectric points and masses - difficult to predict migration patterns
Native PAGE:
Forming first hyrogen bond is unfavorable. If stays in position long enough there is another H bond that will form without energy cost: distruptng cost of initail folding with stabilizing. Entropy cost is distributed throughout the whole molecule. If you fold to form one H bond you've suffered already one entropy cost theres not much entropy to be lost when the nearby H bonds then bond. First little commitment that was the most entropic penalty
Neighboring bonds can help distribute the entropic cost of folding
Rule of thumb: 110 Da per amino acid
No inherent limit to how large proteins can get
(DMSO, DMF)
Non-aqueous polar solvents
Non-covalent molecular forces drive protein structure and binding interactions • Ionic or electrostatic forces • Hydrogen bonding • van der Waals forces • Hydrophobic effect
Non-covalent molecular forces
Electrostatic forces are the strongest - but when you think of number!! There is more van der waals so might contribute more. Hydrophobic effect is not a force its an entropic reaction.
Non-covalent molecular forces stabilizing protein structure
These two reactions supply reactants for each other and will automatically strive towards equilibrium. These reactions provide substrates for each other - which is why we end up at equilibrium
Once there is folded protein available, there will also be a back reaction
-Energetically allowed regions are dictated by steric constraints -Values of φ and ψ are not independent of each other (some value pairs are very common) -Energetically favorable conformations underlie the secondary structure of the peptide backbone Combination of the two angels hat needs to fit
Only a fraction of the possible Φ and Ψ dihedral angles are energetically favorable
Derivatives can be separated by high-pressure liquid chromatography (HPLC) and identified.
PTH-derivatives of amino amides can be separated and identified
Parallel β-strands are more distant in the primary sequence
Parallel β-strands
Amyloids are large β-sheet structures consisting of multiple packed sheets and involving very large numbers of β-strand peptides
Parallel β-strands can form highly stable amyloids associated with neurodegeneration
Polar amino acids are important hydrogen bond donors and acceptors -serine -threonine -Asparagine -Glutamine Hydrogen bonds only form if donor and acceptor bonds are correctly aligned.
Polar amino acids are important....
-linear polymers of acrylamide cross-linked by bisacrylamide - controlled polymer density and pore size
Polyacrylamide gel electrophoresis (PAGE):
Separation of information storage and enzymatic activity Stability over wider range of environments Increased variety of functional groups Cofactors for further functionality
Possible evolutionary benefits of a DNA/protein world
Potassium channels use helix dipole moments and hydrophobic pore residues for selectivity 1. Dipole moment of pore helices stabilize K+ in vestibule 2.Aromatic-hydrophobic interactions create a spring-tensioned selectivity filter
Potassium channels
Can take an unfolded tail and template it to form right base pair and orientation and you get others and others The prion conformation may form spontaneously at low frequency or need another protein to adopt the infectious fold.
Prions become infectious because they template the conversion of more prions
-Proline hydroxylation is a posttranslational modification (uses Vitamin C).
Proline hydroxylation
Solvents, chaotropes, and detergents can solubilize hydrophobic areas whereas Heat denaturation, pH changes, foaming often leads to aggregation of hydrophobic areas Detergents solubilze hydropbobic and covers hydrophobic areas and keeps it soluble - when you remove solvents they can fold correctly again Usually yellow is in inside and urea allows hydrophobic to come on the outside. And If you heat something up enough you can have hydrophobic on outside but they all stick together
Protein denaturation exposes the hydrophobic core
Protein disulfide isomerase helps proteins achieve the correct disulfide bonding in the ER Enzymes that promote protein folding are called chaperones. Lowest energy in the fold is coded in amino acid*** Protein disulfide isomerases do this!! They allow protein to sort it self out (if incorrect disulfide bonds in higher energy states) PDI is an even better catalyst: breaks disulfide bonds and allows them to form their idealized more stable form.
Protein disulfide isomerase
The folding of the peptide chain decreases entropy. The release of water molecules from the salvation shell increases entropy. Creating order and disorder; multiple things happening Two entropic consderations: huge loss of entropy(-) bc now protein is very figid where used to be free but on other hand yay hydropbobic effect: freed up more water so yes + entropy. NET: - loss of entropy
Protein folding involves both loss and gain of entropy
polymer (20+ types of monomers) flexible backbone abundant intra-molecular interactions large variety of secondary structures can be stable over large range of temperatures and pH's (The potential to encode structural information differs strongly between biomolecules)
Proteins
Psi angle is between carbon carbon. Phi is between carbon nitrogen **NOT between peptide bond(resonance) The torsional angles φ and ψ are the major structure determinants of the backbone. φ and ψ are also sterically constrained. Bond rotation along the protein backbone involves mainly the Cα bonds
Psi vs Phi angle
polymer (4 types of monomers) flexible backbone abundant intra-molecular base pairing large variety of secondary structures backbone sensitive to hydrolysis (The potential to encode structural information differs strongly between biomolecules)
RNA
Example: tRNA** Nucleotide composition encodes structural information In addition, base pairing also allows templating for inheritance
RNA can base-pair with itself
No protein needed for this reaction RNA-based "enzymes" = ribozymes
RNA provides the scaffold for orienting RNA ends during splicing and performs the reaction
RNA is the only biomolecule that can act as template and efficient enzyme
RNA world hypothesis
Stopped-flow apparatus: Solution 1:low pH for unfolded Solution 2: buffer at physiological pH Push solutions together and then the reaction starts and so you watch reaction and stop the flow at the light source and then you watch the reaction Solution 1: protein + denaturing agent Solution 2: perfect fixings of good folding environment Start clock and mix and what immediately what will happen is folding. Youre shining a light (left and right dichromatic) so can pick up ellipcitiy of molecules at real time
Rapid measurements are required to measure protein folding
Reaction constants can be determined from the initial rate of the reaction Once reaction gets underway the overall rate is a combination of forward and back reactions We have to measure this before any back reaction happens so you measure initial rates and measure as quickly as possible as soon as the reaction starts to hopefully ignore any back reactions You can use initial reaction rate to help determine the k value. In the first milisecond you can somehow measure the rate bc you know the concentration and boom you have rate constant. Becomes difficult after couple seconds of reaction running.
Reaction constants can be determined from.....
Dissolved protein will unfold until the equilibrium concentrations are reached that are set by the reaction conditions. Note: This is a kinetic experiment : Concentrations change over time until equilibrium is reached. (x-axis: time) Ratio will stay stable dictated by equilibrium constant If start with folded some will unfold until equal reaction rate
Reactions strive towards equilibrium
Dictates possible paths to end up in final energy minimum N = native state, global energy minimum Levainthal paradox: doesn't make since because therye a gradual evolution/intermediates. **know X vs Y axis** Levinthal: Idea is that there is an infiinite possible of wrong confirmations and one right one Idealized: each intermediate is more stable and will naturally get into folded state - gradual Rugged energy: ellaboration of idealized, more complicatied not one easy way to fold A to B. We also have problem of stability (valleys which proteins can get stuck in)
Realistic folding funnels resemble a rugged landscape with multiple metastable minima
(DTT, β-Mercaptoethanol) Reducing agents only destroy disulfide bonds and disrupts tertiary structure
Reducing agents
Proved that removing urea and B-metacaotothanol allowed it to reform. PRVOED: refolding ability is encoded in protein bc all the changed was the presence of the two Now question is how do the cysteins find eachother: something that enables them to go to their lower energy state. He let disulfide bonds form and then removed and then you get scrammbled state. FOLD FIRST THEN DISULFIDE BONDS ARE LOCKED IN THAT STATE and can form anywhere (left) Then showed that right is lower energy state: added trace of B-mercaptoethanol to break the scrammbled bonds and then reorganized to the correct native state Once removed: refold automatically into native state If you reoxidized in precence of urea and then remove: ideal disulfide bonds will happen in certain environments - if you have urea you;; have different disulfide bidges form (metastable confirmation) and locked in** and then ifyou add trace of B-ME it will break the less ideal ones and the correct ones will form on its own. **things can refold on their own and
Refolding of ribonuclease A
Rates are same at equilibrium You can also use kfold/kun to figure out Kf
Relationship between kinetics and equilibrium
Unfavorable changes in entropy can be overcome by favorable changes enthalpy, and vice versa.
Release of heat and change in entropy decide whether a reaction occurs spontaneously
Repeated prolines support formation of an extended polyproline II helix - Extended conformation - Twist due to proline ring Makes a much more tightly packed helix, a more narrow helix/elongated More common in cis so much more common in turns
Repeated prolines
You ccan change amino acids which will affect how easily something will fold Start in unfolded: release free energy and new less stable state but folding path (transition state) is still the same (LEFT) RIGHT: makes folding harder because TS is higher, so blue reaction will go slower bc needs activation energy but is also less stable Can affect stability and transition state**
Residues can independently affect the folding path and protein stability
SDS-PAGE is used to estimate protein size Some proteins migrate slower than predicted: - proteins with many negatively charged amino acids - highly phosphorylated proteins (PO42-) Negative charges interfere with SDS binding and full denaturation.
SDS-PAGE is used to estimate....
Chameleon sequences can assume both α-helix and β-strand conformation depending on the packing environment
Secondary structure can be context-dependent
negative: aspartate and glutamate Aspartate and glutamate side chains act as H+ donors (acids) positive: lysine and arginine Lysine and arginine side chains act as H+ acceptors (bases)
Several amino-acid side chains are charged at physiological pH ranges (pH ~5-8)
One value for when protein is folded and all of these properties change as the protein unfolds.
Several parameters change as proteins unfold
SH2 domain= able to really nicely bind to a phosphorylated tyrosine in the context of different amino acids/neighboring sequences. Specific to the neighboring amino acids, youre not actually changing the structuer itself just the surface and specifiity
Small sequence modifications of domains preserve function while creating specificity
We can shine light to see this: X ray crystalography only if this is highly structured. If not you get scattering. If disordered its moving around, capturing different snapshots to see different conformations
Small-angle X-ray scattering (SAXS) provides insight into the structure of disordered regions
Solvation by water molecules shields charges from each other Have to be in right orbital position for this to work** - cannot be kinked Maintaining the dipoles oriented toward the charge dissipates the electrostatic energy, leading to the high dielectric constant of water.
Solvation by water molecules
The primary amino acid sequence can be used to predict the likelihood of different secondary structures. Computer can take all of combinations and notice a pattern to see which pattern of amino acids will become B sheets and alpha helices
Some amino acid combinations are more likely to form α-helices or β-sheets
Disordered regions are a very common feature of proteins involved in interactions with RNA or DNA and in signaling complexes. Becomes stable upon binding which is what promotes the binding Disordered regions are a very common feature of proteins involved in interactions with RNA or DNA and in signaling complexes.
Some peptide sequences only assume a stable structure in conjunction with a binding partner
Strategies: Increased packing density of the hydrophobic core Increased volume of the hydrophobic core Networks of salt bridges on the surface
Some proteins are extremely stable at high temperatures
The N- and C-terminal regions of proteins are often inherently disordered. Histone tails: no structure. For crystalization to work you need to get all proteins in same confirmation. Tails are not captured in crystal structure = natively disordered regions Very rare that a domain would start right at the beginning of the protein so N and C are inherently disordered often
Some regions of proteins are natively disordered i.e. they will not assume a fixed structure
Virus uses this to cause cancer by taking a gene from organism: virus promotes kinase which allows for growth which leads to cancer
Src kinase is an example of a cellular gene captured by a virus to become an oncogene
B can end up at local minimum and get stuck there bc needs energy to get up and get over hump to get to global minumum Easiest to think of folded and unfolded but there can be more metastable confirmations (arrows): where they get stuck in for a little to get to more stable conformation.
Stable protein folding states are local or global energy minima in the folding funnel
Different phases of folding First step = get out of water as quickly as possible Intermediate folding state which is much slower (side chains not settled) Final: protein tries to optimize H bonding Within miliseconds: hydrophobic collapses (nonpolar things on inside is really large driving force for folding) and then we start to form alpha helices then we form vander waals and salt chains and tertiary structure but takes longer
Stopped-flow measurements indicate that folding occurs at several timescales
Doesn't tell us about order just total weight. If we want to know about order we need to put into a second mass spec and break it in collision cell and mostly break at peptide bonds**** and now can analyze fragments Basically doing MS two times in a row. We have isolated them and send through mass spec and doing light ionization:we just want to give charge not break protein apart and now we can effectively identify mass. If we know protein were looking for is 1000 daltons and then we can extract that and put into mass spec again and looking at one interested in and break again
Tandem mass spectrometry
The 2'-OH can react intramolecularly The reactivity of the 2'-OH group makes RNA much less stable than DNA. Basic conditions allow for nucleophilic attack and get cyclic phosphate intermediate = back bone breaks. The extra OH group is what goes this
The 2'-OH group can participate in reactions
This ignores intermediate states Saying protein will try all of these protein patterns before hits perfect form. Point is that folding will never happen** idea that is a protein tries one and then another from scratch. But there could be one part that folds and then another part and another.
The Levinthal Paradox
The Levinthal paradox ignores the fact that individual conversions can lower the free energy
The Levinthal paradox ignores...
-Domains are relatively rigid folding units: good for structure and to provide binding surfaces -Enzymatic conversion of substrates to products requires flexibility and changes in the conformation of the binding cleft Domains are widely used: by having multiple create flexibility in the protein - what enzymes use a lot. If you put two domains against each other they can create active sites where things are happening Rigid = good for binding sites
The active site of enzymes typically lies at the interface of two domains
The amount of fluorescence quenching indicates what fraction of molecules are unfolded
The amount of fluorescence quenching indicates....
Prion proteins: this changes confirmation and dirfered region forms really stable beta sheet structure (similar to alzhymers) and make fibrils and once you have one of those formed it acts as a template to make other proteins do the same. This sually doesn't happen: for this to form = massive massive barrier. Unlikely it will be in exact postiion to do this but one it does its super stable. Left = normal global minimum Right = way way way further down and makes a super stable structure
The disordered N-terminal tail of human prion protein folds into a stable structure in the prion
Entropy bad enthalpy good but enthalpy outweighs the bad and will fold This means that even small changes in protein structure can tip the balance between folding and unfolding.
The energetic gain of bond formation (∆H°) only barely compensates for the loss of entropy (∆S°)
When K+, DeltaG is negative
The free energy can also be determined from changes in enthalpy and entropy
deltaG lower bc they have OH group (Try, Thr, Ser) Larger the hydrophbic side chain the higher the hydrophobic effect ∆Gtransfer= free energy released to go from exterior of protein (water) to interior (ethanol)
The hydrophobic effect increases for larger hydrophobic side chains
pI = the pH at which a molecule has no net charge.
The isoelectric point (pI)
∆∆Gº = difference in deltaG values, changes should be positive so final should be a higher deltaG than intial which would make sense bc if we mutate its less stable so DeltaG would increase
The mutation of individual amino acids can have a large effect on the free energy of folding
Unfolded protein exposes side chains to surrounding solutions Folded protein would bury hydrophobic residues All nonpolar stuff goes inside bc afraid of water
The oil drop model of protein folding
The pH indicates the concentration of free H+ ions of a given solution pH = −log10 [H+]
The pH indicates
The pKa provides a measure for the amount of protonation at a given pH of the solution If the pKa is lower than the pH, the functional group is mostly deprotonated. If the pKa is higher than the pH, the functional group is mostly protonated.
The pKa provides a
-Delocalization of electrons is energetically favored - Double bonds can only form when all bonds are in one plane - Because of this, peptide bonds are generally planar Peptide bonds have partcial double bond character so doesn't rotate
The peptide bond rapidly switches between two resonance states
The relative concentrations of folded and unfolded protein are constant at equilibrium Important: this equilibrium is not static; there is constant interconversion between folded and unfolded states but the overall concentrations stay the same
The relative concentrations of folded and unfolded protein are......
States have higher free energy and wanna get rid of it ∆G at equilibrium is 0 The energy driving a reaction towards equilibrium is the release of Gibbs Free Energy (ΔG). A reaction will occur spontaneously as long as ΔG is negative (= energy is released) ΔG changes as a function of the concentrations of reactants and products.
The spontaneous drive towards equilibrium is a form of energy
We can infer change in enthalpy by eqilibirum constant (van't hoff equation) Slope = ∆H
The van't Hoff plot allows the determination of the standard enthalpy (∆H°) of folding
Water-accessible surface is buried at the binding interface ASK
The water-accessible surface provides an estimate of the solvation energy of proteins
The α-helix is stabilized by essentially complete intra-backbone H-bonding and abundant van der Waals forces in the core
The α-helix is a tightly packed structure
Melting temperature (Tm): Temperature at which 50% of the protein is unfolded in equilibrium
Tm
∆Gt(Leu-side chain) = ∆Gt(Leu) - ∆Gt(Gly)
To determine ∆Gtransfer of the side chain:
The conjugated aromatic systems of tyrosine and tryptophan absorb UV light and fluoresce Lower energy light comes out in every direction and so we can measure the florescence. Trp and tyr take up UV light and a fraction of the light they give up (lower energy) which we can detect. Shoot UV into sample and it will shoot other light out
Tryptophan and tyrosine are inherent fluorescent probes to study protein folding
Cysteine exists in two oxidative states: reduced and oxidizing Note: the stability of disulfide bonds depends on the environment! Cytoplasm is a reducing environment: S-S bonds are unstable unless shielded. ER/Golgi is an oxidizing environment: disulfide bonds are stable.
Two cysteines can form a disulfide bond
Two-dimensional gel electrophoresis separates proteins by isoelectric focusing and SDS-PAGE Allows separation of very complex protein mixtures (e.g. cell extracts).
Two-dimensional gel electrophoresis
Harder to pull then easier then harder then easier. As your pulling domains are unfolding. As soon as you remove force it will FOLD BACK bc domains are indepdendently folding
Unfolding of the immunoglobulin (Ig) domains may serve as buffer against titin breakage
titin = As you extend you don't want to pull it apart too far so TITIN prevents muscles from separating too much: provides tensile strength to muscles You chemically anchor proteins to beads and since working with such tiny elements they respond to energy of light and when you shine one laser on one bead its trapped within the laser light. Now you can pull other bead and you can measure about of force using to try and get out of this
Use of laser tweezers to analyze the tensile strength of individual titin molecules
-Vitamin C deficiency causes scurvy because of defective collagen.
Vitamin C deficiency
The partial negative charges of the free electron pairs of nitrogen or oxygen support H-bond formation. O-H-O or O-H-N = H bond
Water forms hydrogen bonds with itself and with other compounds
Western blotting uses antibodies to detect a specific protein
Western blotting uses
It will unfold to some extent until a point its at equilibrium bc it is dictated by the constant In ANY environment there needs to be some unfolding in order to achieve equilibrium
What would happen if I dissolved a (100% folded) protein crystal in water?
This system is also initially not in equilibrium. Doesn't matter where you start youll get to same equilibrium Equilibrium tells us nothing about how long it takes. Just that in a certain amount of time it will get to equilibrium
What would happen if I took a fully unfolded peptide chain and allow it to fold?
Protein that doesn't have tryptophan or tyrosine
When would flurescence quenching not work?
Where would the equilibrium lie if the rate constants were the same? Would lie in middle** if rate constants were the same Note that the rate constants for folding and unfolding (or any forward and back reaction) are generally different.
Where would the equilibrium lie if the rate constants were the same?
Normally 35 and then start adding trypsin (cuts R and K), should be 17.5 and 17.5 but this is around 22 each. Should be a homodimer but maybe one is less dense (17.5 in terms of weight but looks like 22 so moves slowly bc spread out). Possibility that this goes to 10 so there could be another 6.25 and 6.25 domain that isnt shown so there are 2 domains Another is that says 35 but in actuallity there is a 22.5 and a linker of 5 and then you have two 7.5s and then you would be able to see
You purify a 35-kD protein that you suspect helps convey a receptor-tyrosine kinase signal to the nucleus. In an effort to determine its domain structure, you subject the protein to limited proteolysis. You observe the following pattern: Schematically draw two possible domain structures that are consistent with this pattern.
Dissociation of SH2 and SH3 domains allosterically changes the structure of the active site. Loss of α-helix makes tyrosine in the activation loop accessible for phosphorylation and c-Src activation Two other domains which regulate kinase SH3 domain and SH2 domain that binds to a phosphoryalted tyrosine and now locked in an inactive state. When this gets activated you get a dephosphorylation Src activator = allosteric regulater then starts chain reaction which allows to unfold and alpha helix is no longer blocking kinase so now open and can activate things
c-Src kinase consists of four interacting domains that link activator binding to kinase activation
Proline is the only amino acid that energetically allows the peptide bond to exists in the cis conformation (ω = 0) at significant levels. Proline has more opportunities for cis compared to other
cis and trans states of proline
Evolutionary problem: Structural information is usually not directly heritable from protein to protein
evolutionary problems of proteins
The charge state of histidine changes at close to neutral pH Alterations in the charge state of histidine are a very common way to control binding interactions and provide a key mechanism for catalysis. Good residue for giving and accepting protons
histidine
A metastable conformation may be become more and more difficult to exit as hydrogen bonds and vdW interactions adjust: kinetic trapping Depending on the folding path, proteins can be trapped in a metastable conformation Local minimum can get lower and lower but still not at optimal
kinetic trapping
The hydrophobic effect is major driving force of protein folding A large part of protein folding is dependent a lot on where the hydrophobic amino acids are Could just be one protein: hydrophobic effect is super significant driving fore
major driving force of protein folding
MS separates proteins and protein fragments based on the mass/charge ratio MS = very good balance: able to weigh the weight of the amino acids Peptide gets ioned (charged) and ow subject to electric field and get acceleration: then you put magnets to force ion to cchange its path. If ion is too heavy to take corner it will crash in the side/small ones also will crash into side and only if it has exact right weight will it make right curve and hit detector. Each time you can change field and scan different weights anddetermine which peptide wights we have. OR can measure how long it takes: heaverier atom takes longer. 1.Ionize: give charge and break apart randomly 2.Electromagnetic accelerator: put in magnetic/electric field 3.Detector
mass spectrometry
Because of steric constraints, the peptide bond is usually in the planar trans configuration Usually things are trans: R groups poking out in different directions cis configuration would create a steric clash between neighboring side chains
peptide bond configuration
The backbone sugar of DNA and RNA differs by one hydroxyl-group 2'-OH makes RNA polymer more flexible 2' oxygen can act as functional group for chemical reactions
ribose vs deoxyribose
Temp changes: as temp goes up entropy is favored so will unfold
temperature changes
At low temperatures many of these mutants are still stable but the unfolding temperature is lower — this is the basis of temperature-sensitive mutations ex: Almost all of these mutations make lysozyme more sensitive to temperature
temperature-sensitive mutations
Dimerization would be an example of 2nd order We don't just stop at equilibirum - we have to worry about the back reaction. As soon as you go away from initial rate Two ways to express rate: imagine no products 0 Fs and one second later there are 10 Fs and the rate would be (10-0/1-0) so rate would be 10 Rate = change in molarity per second and is the same if looking at change in product over time and if you do the negative change in reactants over time --- you get the same value Rate = reactant concentrations x rate constant
the kinetics of a reaction are described by the rate law
Physical systems want energy below zero (lowest energy state) As theyre far apart not as many forces, there is a sweet spot in the middle
van der Waals forces are strongly distance-dependent
The protein core is stabilized by many close packing interactions between hydrophobic side chains.
van der Waals forces become significant with larger numbers of interactions
van der Waals interactions are attractive over short distances because of electron fluctuations -charge fluctuations induce complementary fluctuations nearby -fundamentally attractive -transient and weak -vdW radius depends on size of the electron cloud Example: van der Waals contact between two overall uncharged O2 molecules VDW = energy from electron cloud moving around and will influence neighboring electron clouds - not charged just electrons around them **creates small but consistent force
van der Waals interactions
a chain of amides and Cα's forms the protein backbone Amino N terminus = first amino acid sequenced Carboxy C terminus = last amino acid sequenced
what forms the protein backbone?
Electrostatic forces, vdW forces and H-bonds govern all levels of protein structure
what governs levels of protein structure
Can be positive or negative (ellipticity): scan across different wavelengths and keep measuring. Alpha helices have very particular absorbance spectra as you increase the wavelength To see folding: Alpha/beta to unfolded which shows change in ellipticity which allows us to detect You need to have trp and tyr in core for floresence but with this all you need is secondary structure** Different molecules have different levels of ellipticity. Look at 222nm and if see ellipticy is showing the protein is becoming a random coil which means its unfolding. Why don't we use the 190 value to look? ASK
α helix and β sheet have characteristic ellipticity profiles across the UV spectrum
Charged or polar = interacts with water
α-helices can have faces with different hydrophobicity
- All NH groups point toward the N terminus - All CO groups point toward the C terminus - Creates macroscopic dipole moment - The end of an α-helix often used to stabilize charges
α-helices have dipole moments
The α-helix is a common secondary structure motif in proteins - Angles create a twist in the backbone - Helical conformation when repeated - 3.6 residues per turn - very strong intra-strand H-bonding - average length: 10 amino acids Side chains sticking out, van der walls forces inside H bond between backbones of nitrogen and carbonyl oxygen
α-helix
The two sides of a β-sheet can interact with different environments The β-barrel can form a binding pocket and is one of the most common folds in enzymes (ex: retinol binding protein, triose phosphate isomerase)
β-barrel
- Extended conformation - Creates a zig-zag pattern when repeated - The alternating torsional angles give β-sheets a pleated structure - Neighboring side chains face opposite sides of the sheet Each R is going in different direction.
β-sheets
Dependence on concrantration is showed by this formula You can find equilbirum by setting deltaG = 0 and rearranging equation
∆G is a function of the relative concentrations of reactants and products and temperature
The law of the conservation of energy requires that all reaction paths between (A+B+C) and ABC have the same ∆G Path reaction takes has to be the same in terms of energy If molecule A+B+C to ABC and that cost - 20 jules and that was -10 and combined would be -30 jules, but if you go all the different pathways would need to add up to 0
∆G is independent of the reaction path
