Major Problem in Modern Biochemistry “The Folding Problem”

Background

Information about the three dimensional structure ofa protein is carried in the amino acid sequence- i.e.the gene. (Important concept)

Early experiments

Anfinsen - thermally denature ribonuclease (no cleavage)-refold the protein to be a functional enzyme

How is the information encoded ?

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Ribonuclease :Reversible folding & unfolding

S-S bonds in brown

Denaturation-studiedby various methodsis reversible 2

Problem:-The Lavinthal paradox

Imagine a polypeptide chain of ~100 residues. Assume each amino acid can exist in 10 conformations. Therefore 10100 conformational states are possible.Each has a different set of thermodynamic propertiesHow can the protein sample each state ?

The number of states is 10 21 times greater than one estimate of the number of atoms in the universe. A protein hasn’t time to sample each conformation. Thus, folding must not be a completely random phenomenon.

So-how can proteins fold ?

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Thermodynamic parameters for the folding of some globular proteins at 25 C in aqueous solution

Second problem:The existence of a single conformation of a system that

can exist in 10100 states is unlikely. Why ?

Because in selecting a single state the conformational entropy

S (conformational) = RlnN is lost.

The unfavorable contributionto G is +RTlnN = 8.314 x 300 x ln 10100 = +574 kJ/mole

So there must be stabilizing influences on H to bring the overallG to a minimum

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First Issue:

What features of protein folding produce large , negative Hor large positive S changes, to compensate for the conformational entropy loss ?

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Internal Interactions - energetically favorable interactions between groups within the folded molecule.

1. Charge-Charge Interactions - occur between positively and negatively charged side chain groups. 2. Internal Hydrogen Bonds - interactions between amino acid side chains that are either good hydrogen bond donors (such as the hydroxyls of serine or threonine) or good acceptors (such as the carbonyl oxygens of asparagine or glutamine) . Though hydrogen bonds are relatively weak, the large number of them can make a considerable contribution to stability.3. van der Waals Interactions - weak interactions between uncharged molecular groups in the tightly packed environment of a folded protein. The contributions of these interactions to the negative enthalpy of folding is diminished by giving up favorable interactions with water via folding .

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Common Errors - One of the most common folding errors occurs viacis-trans isomerization of the amide bond adjacent to a proline residue(see here). Proline is the only amino acid in proteins that forms peptidebonds in which the trans isomer is only slightly favored (4 to 1 versus1000 to 1 for other residues). Thus, during folding, there is a significantchance that the wrong proline isomer will form first. It appears that cellshave enzymes to catalyze the cis-trans isomerization necessary to speedcorrect folding.

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DSC of lysozyme at 3 pH values.

Differential Scanning calorimetry (DSC) is used tomeasure the heat required to raise the temperatureof a sample at a constant rate, compared to areference buffer. If the sample is undergoing aphysical phase transition, some heat goes intocausing the structural change so that more heat isneeded to raise the temperature. T2

ΔH = ΔCpdt T1

where ΔCp is the difference in heat capacitybetween the sample and the buffer .

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Dynamics of Protein Folding

How do chains fold into their native structures in spite ofastronomically large numbers of alternatives ?

Clue: Existence of partially unfolded states

High concentrations of Urea (8M) or guanidine-HCl (6M)leads to completely unfolded states of proteins.

But- Other denaturing conditions lead to small changes in hydrodynamic and optical parameters.

1967:-John Brandts showed that acid and thermally denatures proteins can undergo another transition in guanidine-HCl

Example-Bovine or human -lactalbumins undergo twodifferent conformational transitions when guanidine isadded.

Characterization of different states of protein folding dependson the availability of a spectroscopic method.

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NMR Spectroscopy simplified

Nuclei of some elements have spin and behave like small magnets. The spin states are quantized (take on discretevalues)Protons = 1/2, deuterons = 1, 13C = 1/2 (I is the numberusually used for nuclear spin)

There are 2I + 1 states for each nucleus. In an applied magnetic field, these substates have different energies and can be distinguished.

1H substates can have mI = -1/2 or +1/22H substates can have mI = -1, 0 or +1

The energy of these substates in an applied magnetic (H)field is shown below.

The spin of a nucleus can be flipped according to theequation: E = h. This occurs in the microwave region of the spectrum. Experimentally, either the microwave energy can be fixed and the magnetic field changed or vice versa. Normally the magnetic (H) fieldis swept.

Ene

rgy

Magnetic field

I= -1/2

I = +1/2

E = difference in spin state energies

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The main use of NMR is derived from the fact that the energy levels of a spinning nucleus in a magnetic field depend onthe atomic (electronic) environment.

Different protons for example, absorb at different frequencieswith respect to a reference material:

= (Href -H)/ Href x 106 (ppm units)

H = nucleus of interest

Href = reference nucleus

Spectacular advances in NMR result from the fact that nuclear spins also interact through space. Protons closer than 5 perturbeach other’s spins (This is called the Nuclear Overhausereffect). This interaction allows determination of 3D structurein solution.

> 6000 structures in the data base. Usually a “family” of structuresis the solution for any single protein

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At this pH the protein is in a molten globule state and unfolds bya one-step process.The fraction of unfolding is given by fu =(x-xo)/(xu- xo)where x is the value of the measured parameter, xo is its valuein the absence of urea and xu is its value at high levels of urea.Symbols:, Relative intensity of trp fluorescence at 320/360 nm, increase in 1/P (P = trp fluorescence polarization)

, decrease in the negative ellipticity at 220 nm increase in intrinsic viscosityIntrinsic viscosity and the spectrum and polarization of trp fluorescence reflect the compactness of the molecule, while the220 nm ellipticity reflects secondary structure.

Urea-induced unfolding of bovine carbonic anhydrase B (pH 3).

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The fraction of unfolding is given by fu =(x-xo)/(xu- xo)where x is the value of the measured parameter, xo is its valuein the absence of urea and xu is its value at high levels of urea.Symbols: Relative intensity increase in 1/P (P = trp fluorescence polarization) Increase in I(320)/I(360)x, , increase in signal intensities of aliphatic protonsat 3.17, 2.97, 2.00, 0.86 and 1.38 ppmdecrease in negative ellipticity at 270 nm Decrease in area under high field NMR resonanceFluorescence parameters reflect compactness of the moleculeellipticity at 220 reflects secondary structurearea under high field resonance reflects tertiary structure

Unfolding of bovine carbonic anhydrase B at pH= 7.5

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Spectroscopy shows the following:

The first transition (pH 7.5) shows destruction of the rigidenvironment of the aromatic groups,The second transition shows destruction of the secondarystructure

The absence of rigid tertiary structure and the presence of secondary structure lead to a model of the intermediatestate as possessing unfolded, non-compact moleculeswith local secondary structure.

The state is termed the “molten globule”

Properties of the molten globule

I-Compactness . For -lactalbumin, the hydrodynamicradius is ~15% greater than the native state and the volumeis 50% greater than the native state.The fully unfolded molecule (with S-S bonds) has ahydrodynamic radius increased by 49% from the native stateand a volume increased by a factor of 3.3 from the native state.

II. Presence of core. Non-polar groups are in contact but notas tightly as in the native protein

III. Secondary structure. Similar to native

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NMR can be used to determine the mobility of protein structure

HD Exchange can be monitored in D2O solutionSome internal H-bonded atoms can exchange quickly.

Why ?

Local unfolding or “breathing”.Either a normally buried group must surface occasionally to appearat the surface or the reagent must permeate to the interior.

k1 k2

Folded open exchange k-1

k1 <<< k-1

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Five hundred MHz proton NMR spectra of guinea pig-lactalbumin in the native (pH 5.4), acid (pH 2.0) andunfolded (in 9M urea) states recorded at 52oC.

The NMR spectrum of the molten globule state is muchsimpler than that of the native protein;the number of perturbedresonances is much smaller, and the overall picture is muchmore similar to the unfolded state.

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Time-dependence of the spin echo amplitudes for the methyl (A) and aromatic (B) protons of carbonic anhydrase B in the native(pH= 7.2) , molten globule (pH = 3.3) and unfolded (8M Urea)states.

The spin-spin relaxation of the methyl groups in the molten globulestate coincides with the unfolded state, while it is quite differentfor the native state. In contrast, the spin echo curves for the aromatic groups are intermediate between those in the native and in the unfolded states. Therefore, intramolecular movements of aromatic side chains are much more hindered in the molten globule than in the unfolded state, although not as hindered as in the native state.

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IV: Native-like structural Organization- NMR studies show that at least some -helices are locatedin their native positions along the polypeptide chain.How is this done:1. Allow protein in the molten globule state to exchange for a given length of time.2. Transform to native state3. Use 2D-NMR to identify N-H protons protected fromexchange in the molten globule B,C helices in -lactalbumin are protected in the acid molten globule state.4. NMR of the molten globule state is much simpler than thenative molecule. Some pronounced resonance at < 1 ppm in thenative form completely disappear in the molten globule.5. Environment of many side chains is much less rigid in themolten globule vs the normal state(Spin-spin relaxation time diminishes)Motions of methyl groups coincide with the unfolded state whilemotions of aromatics is intermediate between native and unfolded

Why is denaturation so widely studied ? It is thought that denaturationpathways are the opposite of folding pathways.

Old idea-the concept of an all or nothing transition to a completelyunfolded state is probably wrong.

The all or nothing transition is probably native molten globule(movement of side chains, destruction of tight tacking)

Then the system goes from molten globule random coilthrough loss of secondary structure.

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Facts about Molten Globule States in Proteins

1. Proteins can be transformed into the MG state by low orhigh pH, by high temperature, by moderate levels ofurea or guanidine HCl, and by the influence of LiClO4 andother salts (i.e. under mild denaturing conditions).

2. Proteins can be transformed into the MG state withouta change of the environment , simply by small alterationsof their chemical structure.

Examples:I. Staphylococcal nuclease with 21 C terminal residues removedII. Point mutants of lambda repressorIII. BPTI with reduced S-S bondsIV. Alpha-lactalbumins after Ca2+ removal.

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Example: Urea induced unfolding of carbonic anhydrase B at pH 3 compared with pH 7.5

Folding pathway. Folding starts with the formation of fluctuating embryos of regions with secondary structure (stabilized mainly by h-bonds), followed by collapse of these regions into an intermediate compact structure that is stabilized mainly by hydrophobic interactions. The final structure is driven by van der Waals/ and other specific interactions.

Major Problem:

Does any of the above happen in cells?

1. Enzymes exist that accelerate cis-trans isomerization in proline residues, and others catalyze S-S bond rearrangement.

2. All cells contains “chaperonins”, which either aid protein folding or prevent protein folding or prevent proteins from associating prematurely with other proteins.

These were discovered as heat shock proteins accumulated after cells were subjected to temperature jumps or other stress.

Some chaperonins prevent improper folding of membrane proteins-or prevent membrane proteins from aggregating.

GroEL is a beautiful molecular machine. The protein to be protected is bound within the central cavity as a molten globule.

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Chaperonins - In addition to the enzymes mentioned previously that assist with proper folding (e.g., cis-trans isomerase for proline and disulfide bond making enzymes), cells have a class of proteins calledchaperonins, which "chaperone" a protein to help keep it properly folded and non-aggregated. Aggregation is a problem for unfolded proteins because the hydrophobic residues, which normally are deep inside of a protein, may be exposed when the protein is released from the ribosome.If they are exposed to hydrophobic residues in other strands, the two strands may associate with each other hydrophobically (to aggregate) instead of folding properly. These proteins were first identified as heat shock proteins , induced by elevated temperatures or other stress. The most thoroughly studied are hsp-70 (70 kD) and hsp-60 (60-kD). The GroEL-ES complex from E. coli is one such chaperone system (hsp-60). It provides a central cavity in which new protein chains can be "incubated" until they have folded properly .

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The best studied chaperonin: GroEL-ES complex from E. ColiGroEL has two rings each with 7 protein molecules. The centerof each ring has the open hole which is accessible to the solvent.

Either cavity can be capped with Gro-ES again a seven memberedring of smaller subunits.

The insulating property of this molecular machine prevents aggregation or misfolding. The process is ATP-hydrolysis driven

An Amazing Molecular Machine25

Hypothetical Model for chaperonin action in Rubiscofolding.

Active dimer (top) can be unfolded (e.g. 8M Urea)to give an unfolded polypeptide. The dimer can also beacid-denatured to give a polypeptide that still retainselements of secondary structure. It is suspected that a common intermediate forms from either of these twostates on removal of the denaturant. This intermediateis labeled Rubisco I. In the absence of chaperonins, dilution of denatured Rubisco leads to precipitation.If cpn -60 (a chaperonin) is present during dilution, abinary stable complex is formed between it and Rubisco I. When cpn10 and MgATP are added to this complex in thepresence of K+ ions, active Rubisco dimers are formed.

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Rubisco-major protein component of chloroplasts, possiblythe most abundant protein in the world. Its function is thekey step in CO2 fixation in the Calvin cycle of photosynthesis.

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Suppression by groE complex of aggregation duringrefolding. Citrate Synthase was denatured, and refolding wasinitiated by 100-fold dilution of the unfolded protein to theindicated protein concentration. The refolding was measuredin the presence and absence of the GroE complex. A 6-fold molar excess of GroE complex over CS was used.

+ GroE

- GroE

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Prediction of Secondary and Tertiary Protein Structure

Investigators have examined the structures found in proteins and tried to relate them to the individual amino acids.

Secondary Structure - Table 6.6 lists the relative probabilities that a particular amino acid will form an -helix, -sheet, and a "turn" inproteins. Note that the top group of amino acids favors -helices, the middle group favors -sheets, and the last group favors turns.

The Chou-Fasman rules for predicting secondary structure of a region of a polypeptide sequence are the following:

1. Any segment of 6 residues or more with an -helix probability of over 1.03, and not including proline or phenylalanine, is predicted to be -helix.

2. Any segment of 5 residues or more, with beta -sheet probability greater than 1.05 (except histidine) ispredicted to be -sheet.

3. Tetrapeptides with an helix probability less than 0.9 and a turn probability greater than a -sheet probability have a good chance of being turns.

The secondary structures observed in the native protein BPTI as predicted .by the Chou-Fasman rules provides exceptionally good agreement between prediction and experiment.

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Hydrogen exchange of individual backbone amide protonsin BPTI followed by 2D NMR methods. The protein wasdissolved in D2O and kept at 36 C for the indicated timesbefore spectra were acquired.The resonances from the amideprotons that disappear are identified on the last spectrumon which they were apparent, using the one letter code for theamino acids.

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Prediction of protein structures

Rose and Srinivasan assume that protein folding is both localand hierarchichal. Local means that each amino acid’s foldingis influenced by other residues nearby in the sequence. Hierarchichal means that folded structures develop from thesmallest structural units and work up to more and morecomplex entities. The program they have developed iscalled LINUS

“Local Independently Nucleated Units of Structure”

The program considers groups of three amino acids in a sequence-for example residues 12,13,1 4 in a group of 50. The initialassumption is that this group will (randomly) adopt one of 4 possible structures helix, sheet, turn or loop (other). The programthen asks whether this assumed “ministructure” is energeticallysuited to the six amino acids on either side. The program thenmoves on to the next (overlapped) set of residues 13, 14, 15 inthis case.

This random selection and testing is carried out many times for theentire protein. Once this is done, the program analyzes all trials,looking for local groups of amino acids that prefer one of the fourconformations 70% of the time. Such groups are held in theseconformations while the program repeats the entire process usingcomparisons of energetic preferences with respect to 12, aminoacids on either side of selected groups of three residues, then 18 amino acids and so on up to 48 residues.

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The results of LINUS for some proteins are shown below:

Actual and predictedstructures of threedomains of intestinalfatty acid bindingprotein

Actual and predictedstructures of a helicaldomain of cytochromeb-562

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