Field Of Folding Research Articles

Folding by Numbers: Primary Sequence Statistics and Their Use in Studying Protein Folding

The exponential growth over the past several decades in the quantity of both primary sequence data available and the number of protein structures determined has provided a wealth of information describing the relationship between protein primary sequence and tertiary structure. This growing repository of data has served as a prime source for statistical analysis, where underlying relationships between patterns of amino acids and protein structure can be uncovered. Here, we survey the main statistical approaches that have been used for identifying patterns within protein sequences, and discuss sequence pattern research as it relates to both secondary and tertiary protein structure. Limitations to statistical analyses are discussed, and a context for their role within the field of protein folding is given. We conclude by describing a novel statistical study of residue patterning in β-strands, which finds that hydrophobic (i,i+2) pairing in β-strands occurs more often than expected at locations near strand termini. Interpretations involving β-sheet nucleation and growth are discussed.

In memoriam: Reflections on Fred Richards (1925–2009)

Fred Richards made a remarkable discovery right at the start of his scientific career. In his year of postdoctoral research (1954–1955) at the Carlsberg Laboratory, Copenhagen, Fred studied the digestion of RNase A (bovine pancreatic ribonuclease A) by subtilisin, a bacterial protease. He found evidence for a surprising intermediate in the digestion process: see below. Later, in his own lab at Yale, Fred characterized the intermediate (RNase S) by separating it into two parts, a 20-residue peptide (S-pep) and a 104-residue protein moiety (S-prot), and he found that enzymatic activity is lost when the two chains are separated. Remarkably, S-pep and S-prot combine spontaneously when they are mixed together and the product is active enzyme. This result1 immediately clarified how peptide hormones might activate their protein receptors. Because S-prot is only weakly folded and S-pep is nearly structureless, whereas RNase S is a stable, well-folded protein, the reaction S-pep + S-prot = RNase S became a standard system for folding studies and helped to open up the study of the mechanism of protein folding.

Volume changes in protein folding: A Modular Approach

Pressure effects of protein conformational transitions, which are manifest via the associated changes in molar volume, remain rather poorly understood, and regrettably so given that they are clearly linked to changes in the magnitude and type of hydration that accompany such transitions. Given that the role of solvent in protein energetics and structural dynamics remains one of the key questions in the field of protein folding, a better understanding of the physical basis for the volume changes that accompany folding reactions could provide direct means to quantify this differential solvation. We have recently begun high pressure studies on the folding of the ankyrin repeat domain of the protein Notch bearing 7 ankyrin repeats (Nank7) and a series of smaller constructs differing is the number of repeats, and/or their sequences. We reasoned that such a system would provide a means of incrementally testing the role of the size of the hydrophobic core, and the importance of the specific amino acid sequence in determining the volume change upon unfolding, ΔVu. We present here a complete P-T equilibrium and p-jump kinetic study of the full-length Nank7 construct, as well as on a number of the smaller constructs. We find that the volume change of the full-length construct depends strongly upon temperature, and that the expansivity of the transition state ensemble is similar to that of the unfolded state, while its molar volume is closer to that of the folded state. Preliminary results on two very small constructs indicate that the total volume change is a linear combination of the volume change associated with the unfolding of each individual repeat.

Continuous Dissolution Of Structure During The Unfolding Of A Small Protein

An unresolved question in the field of protein folding is whether a protein unfolds in a two-state (N↔U) cooperative manner with only two species being populated during the entire unfolding reaction or in a non-cooperative fashion with a continuum of intermediate forms being populated. To make a definitive distinction between the two has been a difficult challenge, because of the difficulty in identifying and quantifying populations of different species present together during the unfolding reaction. Time-resolved fluorescence resonance energy transfer (TR-FRET) method can differentiate and measure selectively the populations of N, U and I forms, if present together. In this method, energy transfer efficiency is estimated by collecting the decays of fluorescence intensity of the donor fluorophore in the presence or absence of an acceptor. When such fluorescence intensity decays are analyzed by the maximum entropy method (MEM), distributions of fluorescence lifetimes are obtained, which can be used to generate a distribution of distances between the donor and acceptor. In this study, a multi-site, TR-FRET methodology coupled to MEM analysis has been used, for the first time, to study the time evolution of the probability distributions of four intra-molecular distances in the small plant protein monellin, as it unfolds starting from the native state. Surprisingly, one distance is seen to increase completely in a gradual manner, while the increase in the other three distances appears to have both discrete and gradual components. Hence, the protein is found to sample many intermediate conformations, characterized by different intra-molecular distances, before unfolding completely. The observed data can be explained by a simple physical model based upon swelling of a homopolymer chain undergoing diffusive dynamics according to the Rouse model.

From the first protein structures to our current knowledge of protein folding: delights and scepticisms

Every breakthrough that opens new vistas also removes the ground from under the feet of other scientists. The scientific joy of those who have seen the new light is accompanied by the dismay of those whose way of life has been changed for ever. The publication of the first structures of proteins at atomic resolution 50 years ago astounded and inspired scientists in every field, but caused others to flee or scoff. That advance and every subsequent paradigm-shifting breakthrough in protein science have met with some resistance before universal acceptance. I relate these events and their impact on the field of protein folding.

Structural Determinants of the Unusual Helix Stability of a De Novo Engineered Vascular Endothelial Growth Factor (VEGF) Mimicking Peptide

Understanding how an amino acid sequence folds into a well organized three-dimensional structure remains a challenge. The interest in protein folding comes from the possibility to predict the protein structure from genome-derived sequence, design proteins with new fold and understand protein misfolding. Peptide helix is a simple model system in which various contributions to helix formation can be dissected and understood qualitatively. Many strategies have been pursued to design peptide helices and notable results have been achieved even with very short sequences, but mainly these methods rely on the use of nonnatural amino acids or introducing constraints. In this paper, we report on the stability characterization, using CD, NMR and MD studies, of a designed, a-helical, 15-mer peptide (named QK), composed only of natural amino acids (sequence AcKLTWQELYQLKYKGI-NH2), which activates the VEGFdependent angiogenic response. The QK peptide shows an unusual thermal stability, whose structural determinants have been determined. These results could have implication in the field of protein folding and in the design of helical structured scaffolds for the realization of peptides for applications in chemical biology. As recently described, the NMR structure of QK in pure water presents a central helical sequence (residues 4–12), which corresponds to the VEGF N-terminal helix (residues 17–25), flanked by Nand C-capping regions. The helical conformation of QK represents an important prerequisite for its biological activity, since the isolated peptide, corresponding to the helix region of VEGF, does not assume a helical conformation and does not have significant biological activity. Interestingly, QK represents one of the very few examples of bioactive helical designed peptides, composed of only natural amino acids. To gain an insight into the molecular determinants of QK helical propensity, we examined the effect of the temperature on the QK structure through NMR and CD analyses. Primarily, the aggregation state of the peptide under conditions identical to those used in the NMR structure determination was confirmed by NMR DOSY experiments (see Supporting Information). The DOSY-derived diffusion coefficient value of 1.98@10 10 ms 1 is consistent with a QK monomer state. QK structure variations upon temperature increase were followed by TOCSY experiments. In the 298– 343 K range only small changes of the backbone chemical shifts were observed (Table 1 Supporting Information). The temperature dependences of Ha chemical shift deviations from the random coil values (DdHa) are reported in Figure 1a. Unusually, the chemical shift index (CSI) analysis indicates that at 343 K the peptide retains at least the 80% of the helix conformation at 298 K and the slight reduction occurs uniformly in 4–12 region (Figure 1a). The thermal behavior was also analyzed by CD spectroscopy which allowed [a] D. Diana, Prof. Dr. R. Fattorusso Dipartimento di Scienze Ambientali, Seconda UniversitC di Napoli via Vivaldi 43, 81100 Caserta (Italy) Fax: (+39)0823-274605 E-mail : roberto.fattorusso@unina2.it [b] B. Ziaco, Prof. Dr. C. Pedone, Dr. L. D. DIAndrea Istituto di Biostrutture e Bioimmagini, CNR, via Mezzocannone, 16 80134 Napoli (Italy) Fax: (+39)081-2534574 E-mail : ldandrea@unina.it [c] Dr. G. Colombo, Dr. G. Scarabelli Istituto di Chimica del Riconoscimento Molecolare, CNR via Bianco, 9, 20131 Milano (Italy) [d] Dr. A. Romanelli Dipartimento delle Scienze Biologiche UniversitC di Napoli “Federico II” via Mezzocannone 16, 80134 Napoli (Italy) Supporting information for this article is available on the WWW under http://www.chemistry.org or from the author: Peptide synthesis, circular dichroism, nmr spectroscopy and molecular dynamic simulations.

Special issue on protein folding: experimental and theoretical approaches

Special issue on protein folding: experimental and theoretical approaches

Refolding a beta-barrel membrane protein

The field of membrane protein folding is relatively new compared to soluble protein folding. This thesis describes spectroscopy investigations of the refolding and dynamics of a β-barrel membrane protein. The amphiphilic, β-barrel outer membrane protein A (OmpA) refolds and inserts directly into a lipid vesicle or micelle from a denatured state in aqueous urea solution. Spectroscopic probes used to study this system are native tryptophans located at positions 7, 15, 57, 102, and 143. Steady-state and time-resolved fluorescence measurements were performed using single tryptophan mutants of full-length OmpA (325 residues) and the truncated, transmembrane domain (176 residues). Both full-length and truncated mutants exhibit similar tryptophan emission lifetimes, suggesting that the transmembrane microenvironment is not greatly perturbed by the presence of the C-terminus. While the microenvironments of folded full-length and truncated OmpA appear similar, the dynamics of refolding at each tryptophan position exhibit subtle differences when the C-terminus is present. Specifically, we observe that tryptophan-102, which faces the pore interior, inserts and folds the fastest while tryptophan-7, which does not cross the bilayer, is the slowest. Fluorescence anisotropy decays also indicate that tryptophan-7 is the most flexible residue compared to the other tryptophans. Temperature studies below the lipid gel-liquid transition temperature have also been performed. In the lipid gel phase, OmpA adsorbs to the surface of the vesicles but contains immediate β-sheet structure upon folding as well as very hydrophobic tryptophan environments. It is still uncertain from ensemble measurements whether this species is a true intermediate. Fluorescence energy transfer kinetics have successfully determined the intramolecular distance between tryptophan-7 and cysteine-175 labeled with a dansyl fluorophore. These results reveal that the barrel ends of OmpA come into contact early in the refolding process and remain close together up to the final assembly of the barrel. We also have evidence that the adsorbed species at low temperatures is not an intermediate in the folding pathway since no energy transfer is observed for this species. These spectroscopic investigations have provided the foundation for further fundamental studies to dissect the molecular mechanism of the folding pathway of OmpA as well as other integral membrane proteins.

Probing Nature’s Knots: The Folding Pathway of a Knotted Homodimeric Protein

The homodimeric protein YibK from Haemophilus influenzae belongs to a recently discovered superfamily of knotted proteins that has brought about a new protein-folding conundrum. Members of the α/β-knot clan form deep trefoil knots in their native backbone structure, a topological feature that is currently unexplained in the protein-folding field. To help solve the puzzle of how a polypeptide chain can efficiently knot itself, the folding kinetics of YibK have been studied extensively and the results are reported here. Folding was monitored using probes for changes in both secondary and tertiary structure, and the monomer–dimer equilibrium was perturbed with a variety of solution conditions to allow characterisation of otherwise inaccessible states. Multiphasic kinetics were observed in the unfolding and refolding reactions of YibK, and under conditions where the dimer is favoured, dissociation and association were rate-limiting, respectively. A folding model consistent with all kinetic data is proposed: YibK appears to fold via two parallel pathways, partitioned by proline isomerisation events, to two distinct monomeric intermediates. These form a common third intermediate that is able to fold to native dimer. Kinetic simulations suggest that all intermediates are on-pathway. These results provide the valuable groundwork required to further understand how Nature codes for knot formation.

Protein folding thermodynamics and dynamics: where physics, chemistry, and biology meet.

As was noted in our recent review 1 the protein folding field underwent a cyclic development. Initially protein folding was viewed as a strictly experimental field belonging to realm of biochemistry where each protein is viewed as a unique system that requires its own detailed characterization – akin to any mechanism in biology. The theoretical thinking at this stage of development of the field was dominated by the quest to solve so-called “Levinthal paradox” that posits that a protein could not find its native conformation by exhaustive random search. Introduction, in the early nineties, of simplified models to the protein folding field and their success in explaining several key aspects of protein folding, such as two-state folding of many proteins, the nucleation mechanism and its relation to native state topology, have pretty much shifted thinking towards views inspired by physics. The “physics”-centered approach focuses on statistical mechanical aspect of the folding problem by emphasizing universality of folding scenarios over the uniqueness of folding pathways for each protein. Its main achievement is a solution of the protein folding problem in principle, i.e. demonstration how proteins could fold. As a result, a “psychological” solution of the Levinthal paradox was found (i.e. it was generally understood that this is not a paradox. after all). The key success of this stage of the field is discovery of the general requirements for polypeptide sequences to be cooperatively foldable stable proteins and realization that such requirements can be achieved by sequence selection. That put the field strongly into the realm of biology (“Nothing in Biology makes sense except in the light of Evolution” (Theodosius Dobzhansky)) The physics-based fundamental approach to protein folding dominated theoretical thinking in the last decade (reviewed in 1-4) and its successes brought theory and experiment closer together At the present stage we seek better understanding of how protein folding problem is actually solved in Nature. In this sense the protein folding field has made a full circle as attention is again focused on specific proteins and details of their folding mechanism. However these questions are asked at a new level of sophistication of both theory and experiment. Understanding of general principles of folding and vastly improved computer power makes it possible to develop tractable models that sometimes achieve atomic level of accuracy. Further, better general understanding of requirement for polypeptide sequences to fold, lead to establishment of direct links between protein folding and evolution of their sequences This development opened an opportunity to employ powerful methods of bioinformatics to test predictions of various folding models, in addition to more traditional tests of models against experiment After all, evolution presents a giant natural laboratory where sequences are designed to fold and function and availability of vast amounts of data certainly calls for its use to better understand folding of proteins at very high resolution. At the same time in vitro experimental approaches progressed to the point that very accurate time- and structure- resolved data are available. A close interaction with experimentalists helps to keep theorists honest by providing detailed tests of theories and simulation results. In this review, which to a great extent reflects the thinking of the author on the subject, we will first summarize basic questions and present simple, coarse-grained models that provide a basis for a fundamental understanding of protein folding thermodynamics and kinetics. Then we will discuss more recent developments (over last five years) that focus on detailed studies of folding mechanisms of specific proteins, and finally we will briefly discuss some outstanding questions and future directions.

Peptide Secondary Structure Folding Reaction Coordinate: Correlation between UV Raman Amide III Frequency, Ψ Ramachandran Angle, and Hydrogen Bonding

We used UV resonance Raman (UVRR) spectroscopy to quantitatively correlate the peptide bond AmIII3 frequency to its Psi Ramachandran angle and to the number and types of amide hydrogen bonds at different temperatures. This information allows us to develop a family of relationships to directly estimate the Psi Ramachandran angle from measured UVRR AmIII3 frequencies for peptide bonds (PBs) with known hydrogen bonding (HB). These relationships ignore the more modest Phi Ramachandran angle dependence and allow determination of the Psi angle with a standard error of +/-8 degrees , if the HB state of a PB is known. This is normally the case if a known secondary structure motif is studied. Further, if the HB state of a PB in water is unknown, the extreme alterations in such a state could additionally bias the Psi angle by +/-6 degrees . The resulting ability to measure Psi spectroscopically will enable new incisive protein conformational studies, especially in the field of protein folding. This is because any attempt to understand reaction mechanisms requires elucidation of the relevant reaction coordinate(s). The Psi angle is precisely the reaction coordinate that determines secondary structure changes. As shown elsewhere (Mikhonin et al. J. Am. Chem. Soc. 2005, 127, 7712), this correlation can be used to determine portions of the energy landscape along the Psi reaction coordinate.

Stabilization of partially folded states in protein folding/misfolding transitions by hydrostatic pressure

In the last few years, hydrostatic pressure has been extensively used in the study of both protein folding and misfolding/aggregation. Compared to other chemical or physical denaturing agents, a unique feature of pressure is its ability to induce subtle changes in protein conformation, which allow the stabilization of partially folded intermediate states that are usually not significantly populated under more drastic conditions (e.g., in the presence of chemical denaturants or at high temperatures). Much of the recent research in the field of protein folding has focused on the characterization of folding intermediates since these species appear to be involved in a variety of disease-causing protein misfolding and aggregation events. The exact mechanisms of these biological phenomena, however, are still poorly understood. Here, we review recent examples of the use of hydrostatic pressure as a tool to obtain insight into the forces and energetics governing the productive folding or the misfolding and aggregation of proteins.

Folding Studies on a Knotted Protein

YibK is a 160 residue homodimeric protein belonging to the SPOUT class of methyltransferases. Proteins in this group all display a unique topological feature; the backbone polypeptide chain folds to form a deep trefoil knot. Such knotted structures were completely unpredicted, it being thought impossible for a protein to fold efficiently in this way. However, they are becoming more common and there are now a growing number of examples in the Protein Data Bank. These intriguing knotted structures represent a new and significant challenge in the field of protein folding. Here, we present an initial characterisation of the folding of YibK, one of the smallest knotted proteins to be identified. This is the first detailed folding study on a knotted protein to be reported. We have established conditions under which the protein can be denatured reversibly in vitro using urea, thereby showing that molecular chaperones are not required for the efficient folding of this protein. A series of equilibrium unfolding experiments were performed over a 400-fold range of protein concentration. Both secondary and tertiary structural probes show a single, protein concentration-dependent unfolding transition, and data are most consistent with a three-state equilibrium denaturation model involving a monomeric intermediate. Thermodynamic parameters obtained from the fit of the data to this model indicate that the intermediate is a stable species with appreciable secondary and tertiary structure; whether the topological knot remains in the intermediate state is still to be shown. Together, these results demonstrate that, despite its complex knotted structure, YibK is able to fold efficiently and behaves remarkably similarly to other dimeric proteins under equilibrium conditions.

Unfolding and Refolding Pathways of a Major Kinetic Trap in the Oxidative Folding of α-Lactalbumin

Alpha-lactalbumin (alphaLA)-IIIA is a major kinetic intermediate present along the pathways of reductive unfolding and oxidative folding of bovine alpha-lactalbumin (alphaLA). It is a three-disulfide variant of native alphaLA lacking Cys(6)-Cys(120) at the alpha-helical domain. Stability and the unfolding/refolding mechanism of carboxymethylated alphaLA-IIIA have been investigated previously by stop-flow circular dichroism (CD) and fluorescence spectroscopy. A stable intermediate compatible with molten globule was shown to exist along the pathways of unfolding-refolding of alphaLA-IIIA [Ikeguchi et al. (1992) Biochemistry 31, 16695-12700; Horng et al. (2003) Proteins 52, 193-202]. We investigate here the unfolding-refolding pathways and conformational stability of alphaLA-IIIA using the method of disulfide scrambling with the following specific aims: (a) to isolate and characterize the observed stable molten globule, (b) to analyze the heterogeneity of folding-unfolding intermediates, (c) to elucidate the disulfide structure of extensively unfolded isomer of alphaLA-IIIA, and (d) to clarify the relative conformational stability between alphaLA-IIIA and alphaLA. Two scrambled isomers, designated as X-alphaLA-IIIA-c and X-alphaLA-IIIA-a (X stands for scrambled), were isolated under mild and strong denaturing conditions. Their disulfide structures, CD spectra, and manners of refolding to form the native alphaLA-IIIA were analyzed in this report. The results are consistent with the notion that X-alphaLA-IIIA-c and X-alphaLA-IIIA-a represent a partially unfolded and an extensively unfolded isomers of native alphaLA-IIIA, respectively. The unfolding-refolding pathways of alphaLA-IIIA are elaborated and compared with that of intact alphaLA. These results display new insight into one of the most extensively studied molecules in the field of protein folding and unfolding.

The HPr Proteins from the Thermophile Bacillus stearothermophilus can form Domain-swapped Dimers

The study of proteins from extremophilic organisms continues to generate interest in the field of protein folding because paradigms explaining the enhanced stability of these proteins still elude us and such studies have the potential to further our knowledge of the forces stabilizing proteins. We have undertaken such a study with our model protein HPr from a mesophile, Bacillus subtilis, and a thermophile, Bacillus stearothermophilus. We report here the high-resolution structures of the wild-type HPr protein from the thermophile and a variant, F29W. The variant proved to crystallize in two forms: a monomeric form with a structure very similar to the wild-type protein as well as a domain-swapped dimer. Interestingly, the structure of the domain-swapped dimer for HPr is very different from that observed for a homologous protein, Crh, from B.subtilis. The existence of a domain-swapped dimer has implications for amyloid formation and is consistent with recent results showing that the HPr proteins can form amyloid fibrils. We also characterized the conformational stability of the thermophilic HPr proteins using thermal and solvent denaturation methods and have used the high-resolution structures in an attempt to explain the differences in stability between the different HPr proteins. Finally, we present a detailed analysis of the solution properties of the HPr proteins using a variety of biochemical and biophysical methods.

Response to Comment on "Force-Clamp Spectroscopy Monitors the Folding Trajectory of a Single Protein"

Science moves forward when new techniques uncover unanticipated results, and the field of protein folding is no exception. Indeed, our force-clamp spectroscopy measurements of the folding of ubiquitin chains ([ 1 ][1]) revealed trajectories that departed from the expected two-state folding reactions

The Folding of Spectrin Domains I: Wild-type Domains Have the Same Stability but very Different Kinetic Properties

The study of proteins with the same architecture, but different sequence has proven to be a valuable tool in the protein folding field. As a prelude to studies on the folding mechanism of spectrin domains we present the kinetic characterisation of the wild-type forms of the 15th, 16th, and 17th domains of chicken brain alpha-spectrin (referred to as R15, R16 and R17, respectively). We show that the proteins all behave in a two-state manner, with different kinetic properties. The folding rate varies remarkably between different members, with a 5000-fold variation in folding rate and 3000-fold variation in unfolding rate seen for proteins differing only 1 kcal mol(-1) in stability. We show clear evidence for significant complexity in the energy landscape of R16, which shows a change in amplitude outside the stopped-flow timescale and curvature in the unfolding arm of the chevron plot. The accompanying paper describes the characterisation of the folding pathway of this domain.

Keeping up with protein folding

Protein and misfolding in the cellular environment has emerged as a major research theme over the past fifteen years. The implications are broad, ranging from the production of recombinant proteins in a soluble and bioactive form to the understanding of the etiology of neurodegenerative diseases. The basic principle enunciated by Afinsen – that all the information required for a polypeptide to reach a proper conformation is contained within its amino acid sequence [1] – remains unchallenged. However, it is now well established that many proteins require the assistance of modulators to adopt a native structure as they emerge from ribosomes, regain their original conformation following exposure to stress, or traffic to their ultimate destination [2]. One of the most extensively characterized group of modulators are molecular chaperones, a ubiquitous class of proteins that help other polypeptides reach a proper conformation or cellular location without affecting rates or becoming part of the final structure. Molecular function by transiently binding hydrophobic stretches of amino acids that should normally be buried within the core of their substrates but have become solvent-exposed due to improper de novo or imposition of cellular stress (e.g., temperature increase). As a result of the formation of chaperone-substrate complexes, interactive species are shielded from each other and from the solvent and on-pathway is facilitated. Three functionally distinct sets of cooperate in the conformational quality control of the proteome. At the heart of chaperone networks are folding chaperones which use conformational changes fueled by ATP hydrolysis to promote the net refolding/unfolding of bound substrates. Archetypal are the DnaK-DnaJ-GrpE (Hsp70-Hsp40) and GroEL-GroES (Hsp60-Hsp10) systems of E. coli. Holding chaperones (e.g., small heat shock proteins) passively stabilize partially folded species until become available for their refolding. In doing so, they alleviate titration of by unfolding intermediates. Finally, disaggregating chaperones such as E. coli ClpB and S. cerevisiae Hsp104 employ ATP-driven conformational changes to solubilize protein aggregates and transfer partially folded species to for subsequent refolding. Foldases embody the second group of modulators. These enzymes accelerate rate-limiting steps along the pathway and include peptidyl-prolyl cis/trans isomerases (PPIases), which catalyze the trans to cis isomerization of peptidyl-prolyl bonds, and thiol-disulfide oxidoreductases, which promote the formation and isomerization of disulfide bridges (e.g., the Dsb proteins of the E. coli periplasm). The final participants in the protein quality control machinery are proteases which degrade irreversibly damaged polypeptides that cannot be rescued by the above pathways. Nevertheless, nature loves to frustrate reductionist efforts and it has recently become apparent that functional lines are often blurred. For instance, PPIases and oxidoreductases can combine chaperone and foldase functions, certain protease components remodel polypeptide substrates and can thus be viewed as chaperones, some proteases have the ability to switch from hydrolase to chaperone function, and there is evidence that certain proteins classified as may exhibit protease activity (see for instance [3-10]). To keep readers abreast of the fast moving field of protein folding, Microbial Cell Factories has commissioned a series of reviews by experts in the topic. The first of these already appeared in January [11]. In Unscrambling and egg: protein disaggregation by AAA+ proteins, Weibezahn, Bukau and Mogk discuss the discovery, functional characterization and mechanism of action of Hsp104/ClpB, two orthologous ATPases that have the unique ability to fragment protein aggregates. The remodeling activity of other AAA+ proteins (e.g., ClpA, ClpX and ClpC) which are best known more for their role in proteolysis is also examined. The authors close their thorough review by presenting enticing evidence for the roles of AAA+ proteins in the propagation of neurodegenerative diseases. In the second paper of the series [12], Miot and Betton review the mechanisms of protein quality control in the periplasmic space of E. coli. After a brief discussion of the biogenesis of proteins destined for export from the bacterial cytoplasm, the authors describe the players involved in the periplasmic folding-degradation decision and how cells sense and respond to extracytoplasmic stress. The second half of the review provides a historical perspective on how the maltose binding protein has served as a useful model to understand the mechanisms of conformational quality control in the periplasm. With forthcoming reviews on disulfide bridge formation and in vitro refolding, it is a great time to turn to Microbial Cell Factories to keep up with protein folding.

Characterisation of the transition states for protein folding: towards a new level of mechanistic detail in protein engineering analysis

The field of protein folding now offers considerable excitement. Comparative studies of the transition-state structures for a series of protein families with analogous structures have helped to uncover the overall rules for protein folding. In addition, new protein engineering experiments that continuously follow the growth of the folding nucleus have started to fill in the missing details.

Protein folding: Web alert

Various genome projects are identifying thousands of new protein sequences every year, which has led to an expanded interest in protein folding. There are two parts to the protein folding problem: the first is predicting the three-dimensional structure of a protein from its amino-acid sequence; and the second — the focus of this Web alert — is to understand how proteins fold. Increased interest in protein folding has been fuelled by the belief that many human diseases (Alzheimer’s and Parkinson’s, for example) are caused by aggregation or misfolding of proteins. The FASEB Society has a valuable essay (aimed at the general public) that describes known diseases related to protein (mis)folding (www.faseb.org/opar/protfold/protein.html). It is possible that the selective pressures that act during evolution function to preserve amino-acid sequences that not only form structures that adequately perform specific biological functions, but also to preserve those that fold quickly enough to avoid aggregation or misfolded states. Although a search for ‘protein folding’ using a search engine like AltaVista has more than 13,600 hits, the number of useful protein-folding resources is limited. A good place to start, to get acquainted with the people in the field, is the comprehensive list of protein-folding groups compiled by Heinrich Roder’s laboratory at Fox Chase Cancer Center (http://www.fccc.edu/research/labs/roder/folding_groups.html), which contains links to more than 80 research teams working on various aspects of protein folding. Several of the scientists listed here have their own web pages, some of which are very creative, and include links to other useful sites. Protein folding is quite different from small-molecule chemical reactions (in which covalent bonds are formed or broken in well-defined transition states), because of the many degrees of freedom allowed a polypeptide chain. In order to understand the complexity of protein-folding processes at the microscopic level, a statistical approach is required. The current theoretical and computational frontline, with respect to protein-folding dynamics, is well described in the Pittsburgh Supercomputing Center’s interactive collection of articles from 1998 (http://www.psc.edu/science/lifeproc.html). In particular, the papers by C.L. Brooks III and P. Kollman are admirable. Vijay Pande’s website (http://www.stanford.edu/group/pandegroup/#research) is also excellent, and includes movies of all-atom molecular-dynamics simulations of protein folding. The Database of Macromolecular Movements (http://bioinfo.mbb.yale.edu/MolMovDB/) is another amusing site to visit. It contains short videos of motions in proteins (including a few examples of folding and unfolding). This site also includes free software tools for protein geometric analyses. In order to place protein folding in its realistic in vivo context, there are additional factors to address. Around 5% of all proteins made in cells (often larger, multidomain proteins) require help from ‘chaperonin’ proteins to fold properly. To learn more about these reactions, visit the Chaperonin Home Page (http://bioc09.uthscsa.edu/∼seale/Chap/chap.html) located at the University of Texas Health Science Center. This web page links to researchers in the field and lists thermodynamic data for a large number of mutants of the most famous chaperonin pair GroEL/GroES. The site also includes a protein-structure gallery and a recent paper alert. 20–30% of all proteins function within cellular membranes. It has not yet been resolved (from either a thermodynamic or a kinetic viewpoint) how such proteins are synthesized and then inserted into the membranes. An excellent resource for this topic is the web pages of Stephen White’s laboratory at UC Irvine (http://blanco.biomol.uci.edu/mp_assembly.html). The general principles of membrane-protein structure and stability, the group’s own research approach, a wealth of interesting links and downloadable key research papers are all available here. In order to perform their biological functions, many proteins coordinate small cofactors such as metal ions. The recently created PROMISE database (http://bioinf.leeds.ac.uk/promise/) provides a wealth of information on these types of proteins (although there is not much on the proteins’ folding properties yet). In addition, this site links to an impressive number of bioinorganic web resources. The field of protein folding is growing at a fast pace, as is the number of researchers that need to keep up with the current literature and news. A perfect way to do this is to attend the Johns Hopkins Protein Folding Meeting, which has taken place each spring for the past five years. This meeting attracts the finest people in protein folding, and the conference covers all aspects of this fascinating field. The program for the 5th Meeting (taking place this month) can be viewed at http://www.jhu.edu/∼ipmb/protein.html. Protein folding is a rapidly expanding field that involves researchers from many disciplines. We expect new web resources to be developed that continue to facilitate discussion and collaboration.