Search for folding initiation sites from amino acid sequence

A crucial event in protein folding is the formation of a folding nucleus, which is a structured part of the protein chain in the transition state. We demonstrate a correlation between locations of residues involved in the folding nuclei and locations of predicted amyloidogenic regions. The average Phi-values are significantly greater inside amyloidogenic regions than outside them. We have found that fibril formation and normal folding involve many of the same key residues, giving an opportunity to outline the folding initiation site in protein chains. The search for folding initiation sites for apomyoglobin and ribonuclease. A coincides with the predictions made by other approaches.     

Detection of initiation sites in protein folding of the four helix bundle ACBP by chemical shift analysis

A simple alternative method for obtaining "random coil" chemical shifts by intrinsic referencing using the protein's own peptide sequence is presented. These intrinsic random coil backbone shifts were then used to calculate secondary chemical shifts, that provide important information on the residual secondary structure elements in the acid-denatured state of an acyl-coenzyme A binding protein. This method reveals a clear correlation between the carbon secondary chemical shifts and the amide secondary chemical shifts 3-5 residues away in the primary sequence. These findings strongly suggest transient formation of short helix-like segments, and identify unique sequence segments important for protein folding.     

Putative aggregation initiation sites in prion protein

Misfolded prion protein, PrPSc, is believed to be the pathogenic agens in transmissible spongiform encephalopathies. Little is known about the autocatalytic misfolding process. Looking at the intrinsic properties of short sequence stretches, such as conformational flexibility and the tendency to populate extended conformers, we have examined the aggregation behaviour of various peptides within the region 106-157 of the sequence of human prion protein. We observed fast aggregation for the peptide containing residues I138-I-H-F141. This sequence, which is presented at the surface of cellular prion protein, PrPC, in an almost beta-sheet-like conformation, is therefore an ideal anchor-point for initial intermolecular contacts leading to oligomerization. We further report that the aggregation propensity of the neurotoxic peptide 106-126 appears to be centred in its termini and not in the central, alanine-rich sequence (A113-G-AAAA-G-A120).     

Folding of peptide fragments comprising the complete sequence of proteins. Models for initiation of protein folding. II. Plastocyanin.

In an attempt to understand the earliest events in the protein folding pathway, the complete sequence of French bean plastocyanin has been synthesized as a series of short peptide fragments, and the conformational preferences of each peptide examined in aqueous solution using proton n.m.r. methods. Plastocyanin consists largely of beta-sheet, with reverse turns and loops between the strands of the sheet, and one short helix. The n.m.r. experiments indicate that most of the peptides derived from the plastocyanin sequence have remarkably little propensity to adopt folded conformations in aqueous solution, in marked contrast to the peptides derived from the helical protein, myohemerythrin (accompanying paper). For most plastocyanin peptides, the backbone dihedral angles are predominantly in the beta-region of conformational space. Some of the peptides show weak NOE connectivities between adjacent amide protons, indicative of small local populations of backbone conformations in the a region of (phi,psi) space. A conformational preference for a reverse turn is seen in the sequence Ala65-Pro-Gly-Glu68, where a turn structure is found in the folded protein. Significantly, the peptide sequences that populate the alpha-region of (phi,psi) space are mostly derived from turn and loop regions in the protein. The addition of trifluoroethanol does not drive the peptides into helical conformations. In one region of the sequence, the n.m.r. spectra provide evidence of the formation of a hydrophobic cluster involving aromatic and aliphatic side-chains. These results have significance for understanding the initiation of protein folding. From these studies of the fragments of plastocyanin (this paper) and myohemerythrin (accompanying paper), it appears that there is a pre-partitioning of the conformational space sampled by the polypeptide backbone that is related to the secondary structure in the final folded state.     

Folding of peptide fragments comprising the complete sequence of proteins. Models for initiation of protein folding. I. Myohemerythrin.

In an attempt to delineate potential folding initiation sites for different protein structural motifs, we have synthesized series of peptides that span the entire length of the polypeptide chain of two proteins, and examined their conformational preferences in aqueous solution using proton nuclear magnetic resonance and circular dichroism spectroscopy. We describe here the behavior of peptides derived from a simple four-helix bundle protein, myohemerythrin. The peptides correspond to the sequences of the four long helices (the A, B, C and D helices), the N- and C-terminal loops and the connecting sequences between the helices. The peptides corresponding to the helices of the folded protein all exhibit preferences for helix-like conformations in solution. The conformational ensembles of the A- and D-helix peptides contain ordered helical forms, as shown by extensive series of medium-range nuclear Overhauser effect connectivities, while the B- and C-helix peptides exhibit conformational preferences for nascent helix. All four peptides adopt ordered helical conformations in mixtures of trifluoroethanol and water. The terminal and interconnecting loop peptides also appear to contain appreciable populations of conformers with backbone phi and psi angles in the alpha-region and include highly populated hydrophobic cluster and/or turn conformations in some cases. Trifluoroethanol is unable to drive these peptides towards helical conformations. Overall, the peptide fragments of myohemerythrin have a marked preference towards secondary structure formation in aqueous solution. In contrast, peptide fragments derived from the beta-sandwich protein plastocyanin are relatively devoid of secondary structure in aqueous solution (see accompanying paper). These results suggest that the two different protein structural motifs may require different propensities for formation of local elements of secondary structure to initiate folding, and that there is a prepartitioning of conformational space determined by the local amino acid sequence that is different for the helical and beta-sandwich structural motifs.     

Coupled folding during translation initiation

The structure of the eukaryotic initiation factor eIF4E bound to a cognate domain of eIF4G and m(7)GDP in this issue of Cell shows that these factors undergo coupled folding to form a stable complex with high cap binding activity that promotes efficient ribosomal attachment to mRNA during translation initiation.     

Folding myoglobin within a sol-gel glass: protein folding constrained to a small volume

The unfolding and refolding reaction of myoglobin was examined in solution and within a porous silica sol-gel glass. The sol-gel pores constrain the protein to a volume that is the same size and shape as the folded native state accompanied by a few layers of water solvation. Denaturants such as low pH buffers can be diffused through the gel pores to the protein to initiate unfolding and refolding. Acid-induced unfolding was hindered by the steric constraints imposed by the gel pores such that more denaturing conditions were required within the gel than in solution to create the unfolded state. No new folding intermediates were observed. Refolding of myoglobin was not complete in millimolar pH 7 buffer alone. Addition of 25% glycerol to the pH 7 buffer resulted in nearly complete refolding, and the use of 1 M phosphate buffer resulted in complete refolding. The role of this cosolvent and salt in disrupting the ordered water surrounding the protein within the gel is discussed in light of the Hofmeister series and entropic trapping via a diminished hydrophobic effect within the gel. These results are consistent with the premises of folding models in which secondary and tertiary structures are considered to form within a compact conformation of the protein backbone.     

Identification of initiation sites for T4 lysozyme folding using CD and NMR spectroscopy of peptide fragments

Using CD and 2D (1)H NMR spectroscopy, we have identified potential initiation sites for the folding of T4 lysozyme by examining the conformational preferences of peptide fragments corresponding to regions of secondary structure. CD spectropolarimetry showed most peptides were unstructured in water, but adopted partial helical conformations in TFE and SDS solution. This was also consistent with the (1)H NMR data which showed that the peptides were predominantly disordered in water, although in some cases, nascent or small populations of partially folded conformations could be detected. NOE patterns, coupling constants, and deviations from random coil Halpha chemical shift values complemented the CD data and confirmed that many of the peptides were helical in TFE and SDS micelles. In particular, the peptide corresponding to helix E in the native enzyme formed a well-defined helix in both TFE and SDS, indicating that helix E potentially forms an initiation site for T4 lysozyme folding. The data for the other peptides indicated that helices D, F, G, and H are dependent on tertiary interactions for their folding and/or stability. Overall, the results from this study, and those of our earlier studies, are in agreement with modeling and HD-deuterium exchange experiments, and support an hierarchical model of folding for T4 lysozyme.     

Folding of the yeast prion protein Ure2: kinetic evidence for folding and unfolding intermediates.

The Saccharomyces cerevisiae non-Mendelian factor [URE3] propagates by a prion-like mechanism, involving aggregation of the chromosomally encoded protein Ure2. The N-terminal prion domain (PrD) of Ure2 is required for prion activity in vivo and amyloid formation in vitro. However, the molecular mechanism of the prion-like activity remains obscure. Here we measure the kinetics of folding of Ure2 and two N-terminal variants that lack all or part of the PrD. The kinetic folding behaviour of the three proteins is identical, indicating that the PrD does not change the stability, rates of folding or folding pathway of Ure2. Both unfolding and refolding kinetics are multiphasic. An intermediate is populated during unfolding at high denaturant concentrations resulting in the appearance of an unfolding burst phase and roll-over in the denaturant dependence of the unfolding rate constants. During refolding the appearance of a burst phase indicates formation of an intermediate during the dead-time of stopped-flow mixing. A further fast phase shows second-order kinetics, indicating formation of a dimeric intermediate. Regain of native-like fluorescence displays a distinct lag due to population of this on-pathway dimeric intermediate. Double-jump experiments indicate that isomerisation of Pro166, which is cis in the native state, occurs late in refolding after regain of native-like fluorescence. During protein refolding there is kinetic partitioning between productive folding via the dimeric intermediate and a non-productive side reaction via an aggregation prone monomeric intermediate. In the light of this and other studies, schemes for folding, aggregation and prion formation are proposed.     

Chaperones and protein folding

Chaperones are centrally involved in the control of protein structure, function, localization and transport. A flurry of scientific activity continues to examine the molecular nature of chaperone-substrate recognition and the role of auxiliary chaperones (cohort proteins) and small molecules that expedite these processes. Chaperones have been implicated in processes as diverse as protein secretion, nuclear transport, thermotolerance, the steroid receptor signal transduction pathway, T-cell receptor and major histocompatibility complex class I and II multimeric assembly and bacterial virulence.     

Origin of unusual phi-values in protein folding: evidence against specific nucleation sites

phi(f)-value analysis is one of the most common methods to characterize the structure of protein folding transition states. It compares the effects of mutations on the folding kinetics with the respective effects on equilibrium stability. The interpretation of the results usually focuses on a few unusual phi(f)-values, which are either particularly high or which are larger than 1 or smaller than 0. These mutations are believed to affect the most important regions for the folding process. A major uncertainty in experimental phi(f)-values is introduced by the commonly used analysis of only a single mutant at various positions in a protein (two-point analysis). To test the reliability of two-point phi(f)-values we used reference data from three positions in two different proteins at which multiple mutations have been introduced. The results show that two-point phi(f)-values are highly inaccurate if the difference in stability between two variants is less than 7 kJ/mol, corresponding to a 20-fold difference in equilibrium constant. Comparison with reported phi(f)-values for 11 proteins shows that most unusual phi(f)-values are observed in mutants which show changes in protein stability that are too small to allow a reliable analysis. The results argue against specific nucleation sites in protein folding and give a picture of transition states as distorted native states for the major part of a protein or for large substructures.     

Kinetic coupling between protein folding and prolyl isomerization. II. Folding of ribonuclease A and ribonuclease T1.

The folding and unfolding kinetics within the transition region were measured for RNase A and for RNase T1. The data were used to evaluate the theoretical models for the influence of prolyl isomerization on the observed folding kinetics. These two proteins were selected, since the folding reaction of RNase A is faster than prolyl isomerization, whereas in RNase T1, folding is slower than isomerization in the transition region. Folding of RNase T1 was investigated for three variants with different numbers of cis prolyl residues. The results indicate that in the transition region the folding rates are indeed strongly dependent on the number of prolyl residues. The variant of RNase T1 that contains only one cis prolyl residue folds about ten times faster than two variants that contain two cis prolyl residues. For both RNase A and RNase T1, the apparent rates of folding and unfolding as well as the corresponding amplitudes depend on the concentration of denaturant in a manner that was predicted by the model calculations. When refolding was started from the fast-folding species, additional kinetic phases could be observed in the transition region for both proteins. The obtained values could be used to calculate the microscopic rate constants of folding and isomerization on the basis of theoretical models.     

Changes of protein stiffness during folding detect protein folding intermediates

Single-molecule force-quench atomic force microscopy (FQ-AFM) is used to detect folding intermediates of a simple protein by detecting changes of molecular stiffness of the protein during its folding process. Those stiffness changes are obtained from shape and peaks of an autocorrelation of fluctuations in end-to-end length of the folding molecule. The results are supported by predictions of the equipartition theorem and agree with existing Langevin dynamics simulations of a simplified model of a protein folding. In the light of the Langevin simulations the experimental data probe an ensemble of random-coiled collapsed states of the protein, which are present both in the force-quench and thermal-quench folding pathways.     

Peptide models of folding initiation sites of bovine beta-lactoglobulin: identification of nativelike hydrophobic interactions involving G and H strands

In an attempt to characterize the early folding events in bovine beta-lactoglobulin (BLG), a set of peptides, covering the flexible N-terminal region and the stable C-terminus beta-core, was synthesized and analyzed by circular dichroism and by nuclear magnetic resonance in water, trifluoroethanol (TFE), and sodium dodecyl sulfate (SDS) below and above the critical micellar concentration. The role of local and long-range hydrophobic interactions in guiding the folding has been investigated. For the peptide fragment covering the more flexible N-terminal region of BLG (beta-strands A, B), where both theoretical predictions and kinetic refolding experiments suggested the formation of non-native alpha-helix, no native long-range contacts were identified, and a helical secondary structure was stabilized only in the presence of 25 mM SDS. At variance, in 50% (v/v) TFE, native, long-range hydrophobic interactions were observed in the peptide covering the core region comprising G and H beta-strands. The side chains involved in these interactions form a nativelike hydrophobic cluster, thus suggesting that the GH region may act as the folding initiation site for BLG. This result is reinforced by the identification, in the urea denaturated BLG, of residual structure located at the level of the GH interface, as evidenced by NMR analysis. These results, in excellent agreement with kinetic, thermodynamic, and cold denaturation folding data, once more underline the utmost importance of the GH region for the stability and folding of BLG. Severe aggregation effects prevented the structural analysis of the peptide covering the EFGH region, indicating that this larger segment does not represent an independent folding domain and that the terminal alpha-helix is necessary for stabilizing the BLG folding core.     

Cotranslational protein folding

The review analyzes the research concerning the folding of proteins in the course of their synthesis on ribosomes. The experimental data obtained for various proteins using various methods give grounds for concluding that a nascent protein largely acquires its spatial structure while still attached to the ribosome, and final folding into the biologically active conformation takes place as soon as the completed protein is released therefrom. Cotranslational folding is characteristic of both bacterial and eukaryotic cells, and appears to be the universal and the most evolutionarily ancient mechanism.     

Sub-microsecond protein folding

We have investigated the structure, equilibria, and folding kinetics of an engineered 35-residue subdomain of the chicken villin headpiece, an ultrafast-folding protein. Substitution of two buried lysine residues by norleucine residues stabilizes the protein by 1 kcal/mol and increases the folding rate sixfold, as measured by nanosecond laser T-jump. The folding rate at 300 K is (0.7 micros)(-1) with little or no temperature dependence, making this protein the first sub-microsecond folder, with a rate only twofold slower than the theoretically predicted speed limit. Using the 70 ns process to obtain the effective diffusion coefficient, the free energy barrier height is estimated from Kramers theory to be less than approximately 1 kcal/mol. X-ray crystallographic determination at 1A resolution shows no significant change in structure compared to the single-norleucine-substituted molecule and suggests that the increased stability is electrostatic in origin. The ultrafast folding rate, very accurate X-ray structure, and small size make this engineered villin subdomain an ideal system for simulation by atomistic molecular dynamics with explicit solvent.