Distinguishing between sequential and nonsequentially folded proteins: implications for folding and misfolding.

We describe here an algorithm for distinguishing sequential from nonsequentially folding proteins. Several experiments have recently suggested that most of the proteins that are synthesized in the eukaryotic cell may fold sequentially. This proposed folding mechanism in vivo is particularly advantageous to the organism. In the absence of chaperones, the probability that a sequentially folding protein will misfold is reduced significantly. The problem we address here is devising a procedure that would differentiate between the two types of folding patterns. Footprints of sequential folding may be found in structures where consecutive fragments of the chain interact with each other. In such cases, the folding complexity may be viewed as being lower. On the other hand, higher folding complexity suggests that at least a portion of the polypeptide backbone folds back upon itself to form three-dimensional (3D) interactions with noncontiguous portion(s) of the chain. Hence, we look at the mechanism of folding of the molecule via analysis of its complexity, that is, through the 3D interactions formed by contiguous segments on the polypeptide chain. To computationally splice the structure into consecutively interacting fragments, we either cut it into compact hydrophobic folding units or into a set of hypothetical, transient, highly populated, contiguous fragments ("building blocks" of the structure). In sequential folding, successive building blocks interact with each other from the amino to the carboxy terminus of the polypeptide chain. Consequently, the results of the parsing differentiate between sequentially vs. nonsequentially folded chains. The automated assessment of the folding complexity provides insight into both the likelihood of misfolding and the kinetic folding rate of the given protein. In terms of the funnel free energy landscape theory, a protein that truly follows the mechanism of sequential folding, in principle, encounters smoother free energy barriers. A simple sequentially folded protein should, therefore, be less error prone and fold faster than a protein with a complex folding pattern.     

Simulation and experiment conspire to reveal cryptic intermediates and a slide from the nucleation-condensation to framework mechanism of folding

There is a change from three-state to two-state kinetics of folding across the homeodomain superfamily of proteins as the mechanism slides from framework to nucleation-condensation. The tendency for framework folding in this family correlates with inherent helical propensity. The cellular myeloblastis protein (c-Myb) falls in the mechanistic transition region. An earlier, preliminary report of protein engineering experiments and molecular dynamics simulations (MD) showed that the folding mechanism for this protein has aspects of both the nucleation-condensation and framework models. In the more in-depth analysis of the MD trajectories presented here, we find that folding may be attributed to both of these mechanisms in different regions of the protein. The folding of the loop, middle helix, and turn is best described by nucleation-condensation, whereas folding of the N and C-terminal helices may be described by the framework model. Experimentally, c-Myb folds by apparent two-state kinetics, but the MD simulations predict that the kinetics hide a high-energy intermediate. We stabilized this hypothetical folding intermediate by deleting a residue (P174) in the loop between its second and third helices, and the mutant intermediate is long-lived in the simulations. Equilibrium and kinetic experiments demonstrate that folding of the DeltaP174 mutant is indeed three-state. The presence and shape of the intermediate observed in the simulations were confirmed by small angle X-ray scattering experiments.     

Reduced alphabet for protein folding prediction

What are the key building blocks that would have been needed to construct complex protein folds? This is an important issue for understanding protein folding mechanism and guiding de novo protein design. Twenty naturally occurring amino acids and eight secondary structures consist of a 28‐letter alphabet to determine folding kinetics and mechanism. Here we predict folding kinetic rates of proteins from many reduced alphabets. We find that a reduced alphabet of 10 letters achieves good correlation with folding rates, close to the one achieved by full 28‐letter alphabet. Many other reduced alphabets are not significantly correlated to folding rates. The finding suggests that not all amino acids and secondary structures are equally important for protein folding. The foldable sequence of a protein could be designed using at least 10 folding units, which can either promote or inhibit protein folding. Reducing alphabet cardinality without losing key folding kinetic information opens the door to potentially faster machine learning and data mining applications in protein structure prediction, sequence alignment and protein design. Proteins 2015; 83:631–639. © 2015 Wiley Periodicals, Inc.     

Solid-phase peptide synthesis and circular dichroism study of chiral β-peptoid homooligomers

Summary. N-alkyl-β-alanine oligomers (β-peptoids) with α-chiral side chains [(R)- or (S)-1-(phenylethyl)amino groups] were synthesized and analyzed by CD spectroscopy. These chiral β-peptoid homomers exhibited chain-length-dependent and solvent-dependent ellipticity, strongly indicating the presence of a secondary structure in solution. The CD behaviour was only slightly temperature-dependent upon heating, as also previously observed for stable α-peptoid helices containing the same type of side chains. Thus, the data presented here comprise the first evidence for a chain length-dependent secondary folding of compounds with this novel peptidomimetic backbone design. In addition, applicability of a novel hyphenated technique, HPLC-SPE-NMR/MS, for analysis of crude SPPS reaction products was demonstrated. Authors’ address: Dr. C. A. Olsen, Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark; Present address: The Scripps Research Institute     

Statistical analyses of protein folding rates from the view of quantum transition

Understanding protein folding rate is the primary key to unlock the fundamental physics underlying protein structure and its folding mechanism.Especially,the temperature dependence of the folding rate remains unsolved in the literature.Starting from the assumption that protein folding is an event of quantum transition between molecular conformations,we calculated the folding rate for all two-state proteins in a database and studied their temperature dependencies.The non-Arrhenius temperature relation for 16 proteins,whose experimental data had previously been available,was successfully interpreted by comparing the Arrhenius plot with the first-principle calculation.A statistical formula for the prediction of two-state protein folding rate was proposed based on quantum folding theory.The statistical comparisons of the folding rates for 65 two-state proteins were carried out,and the theoretical vs.experimental correlation coefficient was 0.73.Moreover,the maximum and the minimum folding rates given by the theory were consistent with the experimental results.     

GroEL Can Unfold Late Intermediates Populated on the Folding Pathways of Monellin

The modulation of the folding mechanism of the small protein single-chain monellin (MNEI) by the Escherichia coli chaperone GroEL has been studied. In the absence of the chaperone, the folding of monellin occurs via three parallel routes. When folding is initiated in the presence of a saturating concentration of GroEL, only 50-60% of monellin molecules fold completely. The remaining 40-50% of the monellin molecules remain bound to the GroEL and are released only upon addition of ATP. It is shown that the basic folding mechanism of monellin is not altered by the presence of GroEL, but that it occurs via only one of the three available routes when folding is initiated in the presence of saturating concentrations of GroEL. Two pathways become nonoperational because GroEL binds very tightly to early intermediates that populate these pathways in a manner that makes the GroEL-bound intermediates incompetent to fold. This accounts for the monellin molecules that remain GroEL-bound at the end of the folding reaction. The third pathway remains operational because the GroEL-bound early intermediate on this pathway is folding-competent, suggesting that this early intermediate binds to GroEL in a manner that is different from that of the binding of the early intermediates on the other two pathways. It appears, therefore, that the same protein can bind GroEL in more than one way. The modulation of the folding energy landscape of monellin by GroEL occurs because GroEL binds folding intermediates on parallel folding pathways, in different ways, and with different affinities. Moreover, when GroEL is added to refolding monellin at different times after commencement of refolding, the unfolding of two late kinetic intermediates on two of the three folding pathways can be observed. It appears that the unfolding of late folding intermediates is enabled by a thermodynamic coupling mechanism, wherein GroEL binds more tightly to an early intermediate than to a late intermediate on a folding pathway, with preferential binding energy being larger than the stability of the late intermediate. Hence, it is shown that GroEL can inadvertently and passively cause, through its ability to bind different folding intermediates differentially, the unfolding of late productive intermediates on folding pathways, and that its unfolding action is not restricted solely to misfolded or kinetically trapped intermediates.     

Pathway of Oxidative Folding of a 3-Disulfide α-Lactalbumin May Resemble Either BPTI Model or Hirudin Model

Pathways of oxidative folding of disulfide proteins display a high degree of diversity and vary among two extreme models. The BPTI model is defined by limited species of folding intermediates adopting mainly native disulfide bonds. The hirudin model is characterized by highly heterogeneous folding intermediates containing mostly non-native disulfide bonds. αLA-IIIA is a 3-disulfide variant of α-lactalbumin (αLA) with a 3-D conformation essentially identical to that of intact αLA. αLA-IIIA contains 3 native disulfide bonds of αLA, two of them are located at the calcium binding β-subdomain (Cys⁶¹–Cys⁷⁷ and Cys⁷³–Cys⁹¹) and the third bridge is located within the α-helical domain of the molecule (Cys²⁸–Cys¹¹¹). We investigate here the pathway of oxidative folding of fully reduced αLA-IIIA with and without stabilization of its β-subdomain by calcium binding. In the absence of calcium, the folding pathway of αLA-IIIA was shown to resemble that of hirudin model. Upon stabilization of β-sheet domain by calcium binding, the folding pathway of αLA-IIIA exhibits a striking similarity to that of BPTI model. Three predominant folding intermediates of αLA-IIIA containing exclusively native disulfide bonds were isolated and structurally characterized. Our results further demonstrate that stabilization of subdomains in a protein may dictate its folding pathway and represent a major cause for the existing diversity in the folding pathways of the disulfide-containing proteins.     

Folding of bovine growth hormone is consistent with the molten globule hypothesis

Previous results from equilibrium and kinetic studies of the folding of bovine growth hormone (bGH) have demonstrated that bGH does not follow a simple two-step folding mechanism. These results are summarized and interpreted according to the "molten globule" model. The molten globule state of bGH is characterized as a folding intermediate which is largely alpha-helical, retains a compact hydrodynamic radius, has packing of the aromatic side chains that is similar to the unfolded state, and possesses a solvent-exposed hydrophobic surface along helix 106-127 that readily leads to association.     

Rapid collapse precedes the fast two-state folding of the cold shock protein

The cold shock protein Bc-Csp folds very rapidly in a reaction that is well described by a kinetic two-state mechanism without intermediates. We measured the shortening of six intra-protein distances during folding by F?rster resonance energy transfer (FRET) in combination with stopped-flow experiments. Single tryptophan residues were engineered into the protein as the donors, and single 5-(((acetylamino)ethyl)amino)naphthalene-1-sulfonate (AEDANS) residues were placed as the acceptors at solvent-exposed sites of Bc-Csp. Their R0 value of about 22 A was well suited for following distance changes during the folding of this protein with a high sensitivity. The mutagenesis and the labeling did not alter the refolding kinetics. The changes in energy transfer during folding were monitored by both donor and acceptor emission and reciprocal effects were found. In two cases the donor-acceptor distances were similar in the unfolded and the folded state and, as a consequence, the kinetic changes in energy transfer upon folding were very small. For four donor/acceptor pairs we found that > or =50% of the increase in energy transfer upon folding occurred prior to the rate-limiting step of folding. This reveals that about half of the shortening of the intra-molecular distances upon folding has occurred already before the rate-limiting step and suggests that the fast two-state folding reaction of Bc-Csp is preceded by a very rapid collapse.     

Evolutionary computer programming of protein folding and structure predictions

In order to understand the mechanism of protein folding and to assist the rational de-novo design of fast-folding, non-aggregating and stable artificial enzymes it is very helpful to be able to simulate protein folding reactions and to predict the structures of proteins and other biomacromolecules. Here, we use a method of computer programming called "evolutionary computer programming" in which a program evolves depending on the evolutionary pressure exerted on the program. In the case of the presented application of this method on a computer program for folding simulations, the evolutionary pressure exerted was towards faster finding deep minima in the energy landscape of protein folding. Already after 20 evolution steps, the evolved program was able to find deep minima in the energy landscape more than 10 times faster than the original program prior to the evolution process.     

Folding of RNase T1 is decelerated by a specific tertiary contact in a folding intermediate.

The replacement of tryptophan 59 of ribonuclease T1 by a tyrosine residue does not change the stability of the protein. However, it leads to a strong acceleration of a major, proline-limited reaction that is unusually slow in the refolding of the wild-type protein. The distribution of fast- and slow-folding species and the kinetic mechanism of slow folding are not changed by the mutation. Trp-59 is in close contact to Pro-39 in native RNase T1 and probably also in an intermediate that forms rapidly during folding. We suggest that this specific interaction interferes with the trans----cis reisomerization of the Tyr-38-Pro-39 bond at the stage of a native-like folding intermediate. The steric hindrance is abolished either by changing Trp-59 to a less bulky residue, such as tyrosine, or, by a destabilization of folding intermediates at increased concentrations of denaturant. Under such conditions folding of the wild-type protein and of the W59Y variant no longer differ. These results provide strong support for the proposal that trans----cis isomerization of Pro-39 is responsible for the major, very slow refolding reaction of RNase T1. They also indicate that specific tertiary interactions in folding intermediates do exist, but do not necessarily facilitate folding. They can have adverse effects and decelerate rate-limiting steps by trapping partially folded structures.     

Incorporating into a Cα Go model the effects of geometrical restriction on Cα atoms caused by side chain orientations

Coarse‐grained Go models have been widely used for studying protein‐folding mechanisms. Despite the simplicity of the model, these can reproduce the essential features of the folding process of a protein. However, it is also known that side chains significantly contribute to the folding mechanism. Hence, it is desirable to incorporate the side chain effects into a coarse‐grained Go model. In this study, to distinguish the effects of side chain orientation and to understand how these effects contribute to folding mechanisms, we incorporate into a Cα Go model not only heterogeneous contact energies but also geometrical restraints around two Cα atoms in contact with each other. We confirm that the heterogeneity of contact energies governs the folding pathway of a protein and that the geometric constraints attributed to side chains reproduce cooperative transitions in folding. Proteins 2013; 81:1434–1445. © 2013 Wiley Periodicals, Inc.     

GroEL assisted folding of large polypeptide substrates in Escherichia coli: Present scenario and assignments for the future

Escherichia coli chaperonins GroEL and GroES are indispensable for survival and growth of the cell since they provide essential assistance to the folding of many newly translated proteins in the cell. Recent studies indicate that a substantial portion of the proteins involved in the host pathways are completely dependent on GroEL–GroES for their folding and hence providing some explanation for why GroEL is essential for cell growth. Many proteins either small-single domain or large multidomains require assistance from GroEL–ES during their lifetime. Proteins of size up to 70 kDa can fold via the cis mechanism during GroEL–ES assisted pathway, but other proteins (>70 kDa) that cannot be pushed inside the cavity of GroEL–ATP complex upon binding of GroES fold by an evolved mechanism called trans. In recent years, much work has been done on revealing facts about the cis mechanism involving the GroEL assisted folding of small proteins whereas the trans mechanism with larger polypeptide substrates still remains under cover. In order to disentangle the role of chaperonin GroEL–GroES in the folding of large E. coli proteins, this review discusses a number of issues like the range of large polypeptide substrates acted on by GroEL. Do all these substrates need the complete chaperonin system along with ATP for their folding? Does GroEL act as foldase or holdase during the process? We conclude with a discussion of the various queries that need to be resolved in the future for an extensive understanding of the mechanism of GroEL mediated folding of large substrate proteins in E. coli cytosol.     

Charge-charge interactions in the denatured state influence the folding kinetics of ribonuclease Sa

Gaining a better understanding of the denatured state ensemble of proteins is important for understanding protein stability and the mechanism of protein folding. We studied the folding kinetics of ribonuclease Sa (RNase Sa) and a charge-reversal variant (D17R). The refolding kinetics are similar, but the unfolding rate constant is 10-fold greater for the variant. This suggests that charge-charge interactions in the denatured state and the transition state ensembles are more favorable in the variant than in RNase Sa, and shows that charge-charge interactions can influence the kinetics and mechanism of protein folding.     

Protein folding

The problem of protein folding is that how proteins acquire their native unique three‐dimensional structure in the physiological milieu. To solve the problem, the following key questions should be answered: do proteins fold co‐ or post‐translationally, i.e. during or after biosynthesis, what is the mechanism of protein folding, and what is the explanation for fast folding of proteins? The two first questions are discussed in the current review. The general lines are to show that the opinion, that proteins fold after they are synthesized is hardly substantiated and suitable for solving the problem of protein folding and why proteins should fold cotranslationally. A possible tentative model for the mechanism of protein folding is also suggested. To this end, a thorough analysis is made of the biosynthesis, delivery to the folding compartments, and the rates of the biosynthesis, translocation and folding of proteins. A cursory attention is assigned to the role of GroEL/ES‐like chaperonins in protein folding.     

Cunning Simplicity of a Hierarchical Folding

Abstract

A hierarchic scheme of protein folding does not solve the Levinthal paradox since it cannot provide a simultaneous explanation for major features observed for protein folding: (i) folding within non-astronomical time, (ii) independence of the native structure on large variations in the folding rates of given protein under different conditions, and (iii) co-existence, in a visible quantity, of only the native and the unfolded molecules during folding of moderate size (single-domain) proteins. On the contrary, a nucleation mechanism can account for all these major features simultaneously and resolves the Levinthal paradox.     

Insertion of foreign epitopes in HBcAg: how to make the chimeric particle assemble

Summary. Hepatitis B core antigen is one of the most promising protein carriers of foreign epitopes of various human and animal pathogens. Chimeric HBcAg particles can be used as effective artificial immunogenes. Unfortunately, not all chimeric proteins are able to be particulated. The dependence of correct or incorrect folding of chimeric proteins on physical and chemical properties of inserts was studied with the help of ProAnalyst, SALIX and QSARPro computer programs. We have found that insertion of amino acids with high hydrophobicity, large volume, and high β-strand index prevent self-assembling chimeric proteins. These factors are most important for the C-termini of inserts. Recommendations for obtaining correct folding of chimeric HBcAg particles have been given. Received August 8, 1999, Accepted September 26, 1999     

Experimental tests of villin subdomain folding simulations

We have used laser temperature-jump to investigate the kinetics and mechanism of folding the 35 residue subdomain of the villin headpiece. The relaxation kinetics are biphasic with a sub-microsecond phase corresponding to a helix-coil transition and a slower microsecond phase corresponding to overall unfolding/refolding. At 300 K, the folding time is 4.3(+/-0.6) micros, making it the fastest folding, naturally occurring protein, with a rate close to the theoretical speed limit. This time is in remarkable agreement with the prediction of 5 (+11,-3) micros by Zagrovic et al. from atomistic molecular dynamics simulations using an implicit solvent model. We test their prediction that replacement of the C-terminal phenylalanine residue with alanine will increase the folding rate by removing a transient non-native interaction. We find that the alanine substitution has no effect on the folding rate or on the equilibrium constant. Implications of this result for the validity of the simulated folding mechanism are discussed.     

Cotranslational Folding of Proteins

We suppose that folding of proteins occurs cotranslationally by the following scheme. The polypeptide chains enter the folding sites from protein translocation complexes (ribosome, translocation machinery incorporated in membranes) directionally with the N-terminus and gradually. The chain starts to fold as soon as its N-terminal residue enters the folding site from the translocation complex. The folding process accompanies the translocation of the chain to its folding site and is completed after the C-terminal residue leaves the translocation complex. Proteins fold in sequential stages, by translocation of their polypeptide into folding compartments. At each stage a particular conformation of the N-terminal part of the chain that has emerged from the translocation complex is formed. The formation of both the particular conformations of the N-terminal chain segment at each folding stage and the final native protein conformation at the last stage occurs in a time that does not exceed the duration of the fastest elongation cycle on the ribosome.