Protein Folding I and II

Protein Folding I and II Sepideh Khorasanizadeh September 2008 [email protected] Material adapted from text books and journal articles

Protein folding g is cooperative p

The thermal denaturation of Ribonuclease. Solution viscosity increase (open square), near-UV CD at 365 nm (open circle), UV absorbance at 287 nm (open triangles). Filled triangle is a second denaturation after cooling to prove reversibility. reversibility pH 2.1, if physiological pH then Tm = 75 degrees C

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Amino acid sequence determines secondary and tertiary structure.

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The decrease in conformational entropy when a protein folds disfavors folding this is compensated in part by energy stabilization through internal noncovalent bonding.

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Details of H-bonding in a typical protein is shown.

Covalend and noncovalent bond energies.

Noncovalent bonds have one to two orders of magnitude weaker energies

Types of noncovalent interactions. q- and q+ represent a fraction of an electron or proton charge.

Molecules may attract one another by noncovalent forces but can not interpenetrate: van der Waals radii determine molecular surfaces. van der Waals radius is the effective radius for closest molecular packing total interaction energy at any distance is the sum of the attraction and repulsion energies.

Van der V d Waals W l radii dii relevant to Proteins

Hydrogen y g bonds are the strongest g and most specific noncovalent bonds

The internal energy of a system includes all forms of energy that can be exchanged via simple physical process and chemical reactions. First Law of Thermodynamics: The internal energy can change only by the exchange of heat or work k with ith th the surrounding. di Th The h heatt evolved l d iin a reaction ti att constant t t pressure iis equall tto th the enthalpy, ΔH. The enthalpy change in a reaction is the energy change of most interest to biochemists. Reversible processes occur always near a state of equilibrium; irreversible processes drive toward equilibrium. q Entropy is a measure of the randomness or disorder in a system. Second Law of Thermodynamics: The entropy of a system will tend to increase to a maximum value.

The free energy change for a process at constant pressure is: ΔG = ΔH – T ΔS

An example of the interplayy of enthalpyy and entropyy

The signs of ΔH and ΔS determine the effect of temperature on processes or reactions

The burying of hydrophobic groups within a folded protein molecule produces a stabilizing entropy increase known as the hydrophobic effect. effect

Favorable free energy of folding is a net result of Th Thermodynamic d i F Forces

Breaking disulfide bonds by 3 commonly used reagents

β-Mercaptoethanol

DTT

Denaturing chemicals

Disulfide bonds stabilize folding off some proteins t i

Amino acids have to sample and settle with acceptable dihedral angles in native structure of a protein

Folding into an alpha helix requires concerted efforts of side chains and backbone interactions

The relative frequency of every amino acid within diff different t secondary d structures t t

Same chain can be found in two different conformation co o at o in tthe e co context te t o of a d different ee tp protein ote architecture

N

[U] KU =

[N]

=

U

fU 1 - fU

ΔG = - R T ln KU Use of the linear relationship in the unfolding transition region: ΔG (denaturant concentration) = ΔG (zero denaturant) – m x denaturant concentration ΔG (zero denaturant denaturant, H2O) = m (Cmidpoint – C)

Folding Paradox - Levinthal's paradox states that there are approximately 1050 possible conformations for a protein, such as ribonuclease (124 residues). If one new conformation could be attempted every 10-13 seconds, it would still take over 1030 years to randomly test all of the possibilities, y yet ribonuclease can completely fold in about a minute. Thus, folding must not be a completely random phenomenon. Pathway Model - The "pathway" model of protein folding is depicted at the left left. Nucleation is critical because it is much more difficult to begin an helix than to extend it. Nucleation may start at a number of points and all of these partially folded structures can be "funneled" by energy minimizations toward the final state. Thus, Levinthal's paradox is averted.

Matthews, Ahern, and van Holde, 3rd ed.

Energy surface for protein conformations Levinthal “golf course” landscape

Funnell with F ith various i paths th iis more realistic.

Refolding and S-S bond formation in BPTI. The intermediate structure may contain both native and nonnative S-S bonds. Interconversion within boxed regions is rapid.

Disulfide Bond Formation - Proteins with disulfide bonds have a built-in advantage if they are denatured with their disulfide bonds intact intact. The intact disulfide bonds eliminate many degrees of freedom associated with denaturation, denaturation so fewer events need to occur to bring about the correctly folded state. This can be verified by removing the disulfide bonds of a protein and then denaturing it. Refolding of this polypeptide occurs, but at a slower rate than when the disulfides are left intact. Interestingly, disulfide bonds not found in the native structure sometimes form during intermediate stages of folding. Also, the folding process can be aided by enzymes that make disulfide bonds.

Cis versus Trans Conformation A factor of 4:1 occurrence in Proline vs. 1000:1 in other th amino i acids id

Common Errors - One of the most common folding errors occurs via cis-trans isomerization of the amide bond adjacent j to a p proline residue. Proline is the only amino acid in proteins that forms peptide bonds in which the trans isomer is only slightly favored (4 to 1 versus 1000 to 1 for other residues). Thus, during folding, there is a significant chance that the wrong proline isomer will form first first. It appears that cells have enzymes to catalyze the cistrans isomerization necessary to speed correct folding.

Time scale of protein motions

The Levinthal 'golf-course' landscape. N is the native conformation. The chain searches for N randomly, that is, on a level playing field of energies energies.

The 'pathway' solution to the random search problem of Fig. 1. A pathway is assumed to lead from a denatured conformation A to the native conformation N, N so conformational searching is more directed and folding is faster than for random searching.

An idealized funnel landscape. As the chain forms increasing numbers b off iintrachain t h i contacts, t t and lowers its internal free energy, its conformational freedom is also reduced.

Dill and Chan, (1997) From Levinthal to pathways to funnels, Nature Structural Biology, 4:10-19

Differentt folding Diff f ldi scenarios. i Th vertical The ti l axis i is i internal i t l free f energy. Each E h conformation f ti iis represented as a point on the landscape. The two horizontal axes represent the many chain degrees of freedom. (a) shows a rugged landscape with hills and traps, folding kinetics is likely multiexponential. (b) shows a landscape in which folding is faster than unfolding. A is a throughway folding path,, whereas unfolding p g chains (p (path B)) must surmount a barrier in order to reach the most stable denatured conformations. (c) shows a landscape in which folding is slower than unfolding. Most folding paths (path A) pass through a kinetic trap, whereas some low-lying denatured conformations are readily accessible from the native state during unfolding (path B).

Chan and Dill, (1998) Proteins 30:2-33.

Funnelscape for a fast folding protein Folding is limited by the rate of meandering downhill downhill.

Champagne glass landscape, to illustrate how conformational entropy can cause "free energy barriers" to folding. The "bottleneck" or rate limit to folding is the aimless wandering on the flat plateau as the chain tries to find its way downhill (From From Levinthal to pathways to funnels) (b) Serpin scenario shows a landscape with a deep kinetic trap on the left (A), which is easily accessible from the open conformations. Chain trapped in this deep local minima anneal to the global minimum (B, (B in the middle) only very slowly. This corresponds to the folding of some serpins such as PAI-1.

Chan and Dill, (1998) Proteins 30:2-33.

Figure 4. A rugged energy landscape with kinetic traps, energy barriers, and some narrow throughway ppaths to native. Foldingg can be multi-state.

Figure 6. Champagne Glass Landscape, to illustrate how conformational entropy can cause free energy barriers to folding. The 'bottleneck' or rate limit to folding is the aimless wandering on the flat plateau as the chain tries to find its way downhill.

Dill and Chan, (1997) Nature Structural Biology, 4:10-19

Figure 5. Moat Landscape, to illustrate how a protein could have a fast-folding throughway process (A), in parallel with a slow-foldingg pprocess ((B)) involvingg a kinetic trap. p

Global distribution of conformations of a polypeptide chain in a random-coil state, a partially collapsed denatured state and a compact non-native state. The different species within each ensemble interconvert rapidly. (b) Chemical structure of an alanine residue and the Ramachandran diagram representing its free-energy free energy surface in a protein environment. The surface features result primarily from steric repulsions between the various atoms and lead to a distribution of conformations in the coil state that corresponds locally to an average over the low-energy regions of the conformational space for each amino acid. The diagram was obtained by taking the natural logarithm of the observed frequency of each pair of main-chain dihedral angles (φ,ψ) in a set of 1000 representative protein structures). The contours are spaced 0.5 units apart, so that each level is a factor of e 0.5=1.6 times more probable than the previous (lower) one.

Dinner et al. (2000) Trends Biochem Sci 25:331-339.

Atomic structures of amyloid cross- spines reveal varied steric zippers Nature 447, 453-457 (2007) Sawaya et al. http://www ncbi nlm nih gov/entrez/query fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list uids=17468747 http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17468747

Amyloid fibrils formed from different proteins, each associated with a particular disease, contain a common cross- spine. The atomic architecture of a spine, from the fibril-forming segment GNNQQNY of the yeast prion protein Sup35, was recently revealed by X-ray X ray microcrystallography. It is a pair of -sheets, with the facing side chains of the two sheets interdigitated in a dry 'steric zipper'. Here we report some 30 other segments from fibril-forming proteins that form amyloid-like fibrils, microcrystals, or usually both. These include segments from the Alzheimer's Alzheimer s amyloid amyloid- and tau proteins, the PrP prion protein, insulin, islet amyloid polypeptide (IAPP), lysozyme, myoglobin, -synuclein and 2-microglobulin, suggesting that common structural features are shared by amyloid diseases at the molecular level. Structures of 13 of these microcrystals all reveal steric zippers, but with variations that expand the range of atomic architectures for amyloid amyloid-like like fibrils and offer an atomic-level hypothesis for the basis of prion strains.

Despite their fundamental similarity, th reported the t d structures t t display di l variations i ti of the basic steric-zipper structure and thereby expand our understanding of amyloid y structure.

Th 8 classes The l off steric t i zippers i

Mechanism of coupled folding and binding of an intrinsically disordered protein Sugase g et al.

Nature 447, 1021-1025 (2007) http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17522630

Protein P t i ffolding ldi and d bi binding di are analogous l processes, iin which hi h th the protein t i ''searches' h ' ffor favourable f bl intramolecular or intermolecular interactions on a funnelled energy landscape1, 2. Many eukaryotic proteins are disordered under physiological conditions, and fold into ordered structures only on binding to their cellular targets. The mechanism by which folding is coupled to binding is poorly l understood, d t d b butt it h has b been h hypothesized th i d on th theoretical ti l grounds d th thatt th the bi binding di ki kinetics ti may be enhanced by a 'fly-casting' effect, where the disordered protein binds weakly and non-specifically to its target and folds as it approaches the cognate binding site7. Here we show, using NMR titrations and 15N relaxation dispersion, that the phosphorylated kinase inducible acti ation domain (pKID) of the transcription factor CREB forms an ensemble of transient enco activation encounter nter complexes on binding to the KIX domain of the CREB binding protein. The encounter complexes are stabilized primarily by non-specific hydrophobic contacts, and evolve by way of an intermediate to the fully bound state without dissociation from KIX. The carboxy-terminal helix of pKID is only partially folded in the intermediate intermediate, and becomes stabilized by intermolecular interactions formed in the final bound state. Future applications of our method will provide new understanding of the molecular mechanisms by which intrinsically disordered proteins perform their diverse biological functions.

Coupled Folding and Binding

Interaction between the pKID domain of the gene transcription factor CREB and the KIX domain of the CREB-binding protein occurs in the cell nucleus to regulate gene expression. By elucidating the three-step binding reaction between pKID and KIX using NMR spectroscopy, Sugase et al. identified four states along the reaction pathway. Initially, the highly disordered, free state of pKID partially populates l t helix h li A (A). ) In the encounter complex with KIX, pKID is tethered by nonspecific hydrophobic contacts in its helix B region( g (B)). The intermediate state is characterized by a specifically bound and largely configured helix A. Finally, in the high Finally high-affinity, affinity bound conformation conformation, both helices are fully structured.

Enzymes decrease the activation energy and facilitate the formation of the transition state Related to the Chaperonin-assisted Protein Folding

Possible functional cycle of the GroEL-GroES Chaperonin

An unfoled protein molecule binds to one end of the GroEL GroEL-ADP ADP complex with bound GroES at the other end. Folding can occur inside the cavity or The cavity may provide an unfolding opportunity to allow refolding.

Further Reading: The Two Families of Chaperonin: Physiology and Mechanism Annu. Rev. Cell Dev. Biol. 23, 115-45 (2007) http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17489689