When life arose from prebiotic molecules 3.5 billion years ago, what came first? Informational molecules (RNA, DNA), functional ones (proteins), or something else? We argue here for a different logic: rather than seeking a molecule type, we seek a dynamical process. Biology required an ability to evolve before it could choose and optimise materials. We hypothesise that the evolution process was rooted in the peptide folding process. Modelling shows how short random peptides can collapse in water and catalyse the elongation of others, powering both increased folding stability and emergent autocatalysis through a disorder-to-order process.

Divalent sulfur (S) forms a chalcogen bond (Ch-bond) via its σ-holes and a hydrogen bond (H-bond) via its lone pairs. The relevance of these interactions and their interplay for protein structure and function is unclear. Based on the analyses of the crystal structures of small organic/organometallic molecules and proteins and their molecular electrostatic surface potential, we show that the reciprocity of the substituent-dependent strength of the σ-holes and lone pairs correlates with the formation of either Ch-bond or H-bond. In proteins, cystines preferentially form Ch-bonds, metal-chelated cysteines form H-bonds, while methionines form either of them with comparable frequencies. This has implications for the positioning of these residues and their role in protein structure and function. Computational analyses reveal that the S-mediated interactions stabilise protein secondary structures by mechanisms such as helix capping and protecting free β-sheet edges by negative design. The study highlights the importance of S-mediated Ch-bond and H-bond for understanding protein folding and function, the development of improved strategies for protein/peptide structure prediction and design and structure-based drug discovery.

The advent of single-molecule force spectroscopy represents the introduction of forces, torques, and displacements as controlled variables in biochemistry. These methods afford the direct manipulation of individual molecules to interrogate the forces that hold together their structure, the forces and torques that these molecules generate in the course of their biochemical reactions, and the use of force, torque, and displacement as tools to investigate the mechanisms of these reactions. Because of their microscopic nature, the signals detected in these experiments are often dominated by fluctuations, which, in turn, play an important role in the mechanisms that underlie the operation of the molecular machines of the cell. Their direct observation and quantification in single-molecule experiments provide a unique window to investigate those mechanisms, as well as a convenient way to investigate fundamental new fluctuation theorems of statistical mechanics that bridge the equilibrium and non-equilibrium realms of this discipline. In this review we have concentrated on the developments that occurred in our laboratory on the characterization of biopolymers and of molecular machines of the central dogma. Accordingly, some important areas like the study of cytoskeletal motors have not been included. While we adopt at times an anecdotal perspective with the hope of conveying the personal circumstances in which these developments took place, we have made every effort, nonetheless, to include the most important developments that were taking place at the same time in other laboratories.

Ankyrin (ANK) repeat proteins are coded by tandem occurrences of patterns with around 33 amino acids. They often mediate protein–protein interactions in a diversity of biological systems. These proteins have an elongated non-globular shape and often display complex folding mechanisms. This work investigates the energy landscape of representative proteins of this class made up of 3, 4 and 6 ANK repeats using the energy-landscape visualisation method (ELViM). By combining biased and unbiased coarse-grained molecular dynamics AWSEM simulations that sample conformations along the folding trajectories with the ELViM structure-based phase space, one finds a three-dimensional representation of the globally funnelled energy surface. In this representation, it is possible to delineate distinct folding pathways. We show that ELViMs can project, in a natural way, the intricacies of the highly dimensional energy landscapes encoded by the highly symmetric ankyrin repeat proteins into useful low-dimensional representations. These projections can discriminate between multiplicities of specific parallel folding mechanisms that otherwise can be hidden in oversimplified depictions.

This chronologue seeks to document the discovery and development of an understanding of oligomeric ring protein assemblies known as chaperonins that assist protein folding in the cell. It provides detail regarding genetic, physiologic, biochemical, and biophysical studies of these ATP-utilizing machines from both in vivo and in vitro observations. The chronologue is organized into various topics of physiology and mechanism, for each of which a chronologic order is generally followed. The text is liberally illustrated to provide firsthand inspection of the key pieces of experimental data that propelled this field. Because of the length and depth of this piece, the use of the outline as a guide for selected reading is encouraged, but it should also be of help in pursuing the text in direct order.

Protein disulfide isomerase (PDI E.C. 5.3.4.1) catalyses the formation of disulfide bonds in secretory or cell-surface proteins during their biosynthesis. PDI activity is associated with the microsomal fraction of the cell and has been found in several mammalian tissues and in developing wheat endosperm.

The embryo and aleurone layer of germinating seeds of the wheat cultivar Galahad showed PDI activity associated with a microsome-enriched fraction of cell homogenates PDI activity in microsome-enriched fractions from aleurone layers of normally germinating seeds was significantly higher than in their abnormally germinating counterparts and diminished as germination proceeded.

Dry embryos and germinating embryos showed less PDI activity than aleurone layers. There was no significant difference in PDI activity between microsome-enriched fractions from embryos of normally and abnormally germinating seeds at 3 or 6 days of germination.

Wheat aleurone PDI was partially purified using gel filtration and had a molecular mass of 110–130 kDa and a monomeric molecular mass of ca. 57 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis.

The HP model is one of the most popular discretized models for attacking the protein folding problem, i.e., for the computational prediction of the tertiary structure of a protein from its amino acid sequence. It is based on the assumption that interactions between hydrophobic amino acids are the main force in the folding process. Therefore, it distinguishes between polar and hydrophobic amino acids only and tries to embed the amino acid sequence into a two- or three-dimensional grid lattice such as to maximize the number of contacts, i.e., of pairs of hydrophobic amino acids that are embedded into neighboring positions of the grid. In this paper, we propose a new generalization of the HP model which overcomes one of the major drawbacks of the original HP model, namely the bipartiteness of the underlying grid structure which severely restricts the set of possible contacts. Moreover, we introduce the (biologically well-motivated) concept of weighted contacts, where each contact gets assigned a weight depending on the spatial distance between the embedded amino acids. We analyze the applicability of existing approximation algorithms for the original HP model to our new setting and design a new approximation algorithm for this generalized model.

We performed genome-wide mutagenesis of C57BL/6J mice using the mutagen N-ethyl-N-nitrosourea (ENU) and screened the third generation (G3) offspring for visual system alterations using electroretinography and fundus photography. Several mice in one pedigree showed characteristics of retinal degeneration when tested at 12–14 weeks of age: no recordable electroretinogram (ERG), attenuation of retinal vessels, and speckled pigmentation of the fundus. Histological studies showed that the retinas undergo a photoreceptor degeneration with apoptotic loss of outer nuclear layer nuclei but visual acuity measured using the optomotor response under photopic conditions persists in spite of considerable photoreceptor loss. The Noerg-1 mutation showed an autosomal dominant pattern of inheritance in progeny. Studies in early postnatal mice showed degeneration to occur after formation of partially functional rods. The Noerg-1 mutation was mapped genetically to chromosome 6 by crossing C57BL/6J mutants with DBA/2J or BALB/cJ mice to produce an N2 generation and then determining the ERG phenotypes and the genotypes of the N2 offspring at multiple loci using SSLP and SNP markers. Fine mapping was accomplished with a set of closely spaced markers. A nonrecombinant region from 112.8 Mb to 115.1 Mb was identified, encompassing the rhodopsin (Rho) coding region. A single nucleotide transition from G to A was found in the Rho gene that is predicted to result in a substitution of Tyr for Cys at position 110, in an intradiscal loop. This mutation has been found in patients with autosomal dominant retinitis pigmentosa (RP) and results in misfolding of rhodopsin expressed in vitro. Thus, ENU mutagenesis is capable of replicating mutations that occur in human patients and is useful for generating de novo models of human inherited eye disease. Furthermore, the availability of the mouse genomic sequence and extensive DNA polymorphisms made the rapid identification of this gene possible, demonstrating that the use of ENU-induced mutations for functional gene identification is now practical for individual laboratories.

The longevity of filarial nematodes is dependent on secreted immunomodulatory products. Previous investigation of one such product, ES-62, has suggested a critical role for post-translationally attached phosphorylcholine (PC) moieties. In order to further investigate this, ES-62 lacking PC was produced, using the Pichia pastoris recombinant gene expression system. Unlike parasite-derived ES-62, which is tetrameric the recombinant material was found to consist of a mixture of apparently stable tetramers, dimers and monomers. Nevertheless, the recombinant protein was considered to be an adequate PC-free ES-62 as it was recognized by existing antisera against the parasite-derived protein. However, subsequent to this, recognition of parasite-derived ES-62 by antibodies produced against the recombinant protein was found to be absent. In an attempt to explain this, recombinant ES-62 was subjected to structural analysis and was found to (i) contain 3 changes in amino acid composition; (ii) demonstrate significant alterations in glycosylation; (iii) show major differences in protein secondary structure. The effects of these alterations in relation to the observed change in immunogenicity were investigated and are discussed. The data presented clearly show that recognition by existing antibodies is insufficient proof that recombinant proteins can be used to mimic parasite-derived material in studies on nematode immunology and vaccination.

We consider two versions of a simple evolutionary algorithm (EA) model for protein folding at zero temperature, namely the (1 + 1)-EA on the LeadingOnes problem. In this schematic model, the structure of the protein, which is encoded as a bit-string of length n, is evolved to its native conformation through a stochastic pathway of sequential contact bindings. We study the asymptotic behavior of the hitting time, in the mean case scenario, under two different mutations: the one-flip, which flips a unique bit chosen uniformly at random in the bit-string, and the Bernoulli-flip, which flips each bit in the bit-string independently with probability c/n, for some c ∈ ℝ+ (0 ≤ c ≤ n). For each algorithm, we prove a law of large numbers, a central limit theorem, and compare the performance of the two models.

α-Synuclein, a major constituent of Lewy bodies (LBs) in Parkinson's disease (PD), has been implicated to play a critical role in synaptic events, such as neuronal plasticity during development, learning, and degeneration under pathological conditions, although the physiological function of α-synuclein has not yet been established. We here present biochemical evidence that recombinant α-synuclein has a chaperone-like function against thermal and chemical stress in vitro. In our experiments, α-synuclein protected glutathione S-transferase (GST) and aldolase from heat-induced precipitation, and α-lactalbumin and bovine serum albumin from dithiothreitol (DTT)-induced precipitation like other molecular chaperones. Moreover, preheating of α-synuclein, which is believed to reorganize the molecular surface of α-synuclein, increased the chaperone-like activity. Interestingly, in organic solvents, which promotes the formation of secondary structure, α-synuclein aggregated more easily than in its native condition, which eventually might abrogate the chaperone-like function of the protein. In addition, α-synuclein was also rapidly and significantly precipitated by heat in the presence of Zn2+ in vitro, whereas it was not affected by the presence of Ca2+ or Mg2+. Circular dichroism spectra confirmed that α-synuclein underwent conformational change in the presence of Zn2+. Taken together, our data suggest that α-synuclein could act as a molecular chaperone, and that the conformational change of the α-synuclein could explain the aggregation kinetics of α-synuclein, which may be related to the abolishment of the chaperonic-like activity.

One of the proposed roles of the GroEL-GroES cavity is to provide an “infinite dilution” folding chamber where protein substrate can fold avoiding deleterious off-pathway aggregation. Support for this hypothesis has been strengthened by a number of studies that demonstrated a mandatory GroES requirement under nonpermissive solution conditions, i.e., the conditions where proteins cannot spontaneously fold. We have found that the refolding of glutamine synthetase (GS) does not follow this pattern. In the presence of natural osmolytes trimethylamine N-oxide (TMAO) or potassium glutamate, refolding GS monomers readily aggregate into very large inactive complexes and fail to reactivate even at low protein concentration. Surprisingly, under these “nonpermissive” folding conditions, GS can reactivate with GroEL and ATP alone and does not require the encapsulation by GroES. In contrast, the chaperonin dependent reactivation of GS under another nonpermissive condition of low Mg2+ (<2 mM MgCl2) shows an absolute requirement of GroES. High-performance liquid chromatography gel filtration analysis and irreversible misfolding kinetics show that a major species of the GS folding intermediates, generated under these “low Mg2+” conditions exist as long-lived metastable monomers that can be reactivated after a significantly delayed addition of the GroEL. Our results indicate that the GroES requirement for refolding of GS is not simply dictated by the aggregation propensity of this protein substrate. Our data also suggest that the GroEL-GroES encapsulated environment is not required under all nonpermissive folding conditions.

Because of the simplicity and regularity of the α-helical coiled coil relative to other structural motifs, it can be conveniently used to clarify the molecular interactions responsible for protein folding and stability. Here we describe the de novo design and characterization of a two heptad-repeat peptide stabilized by a complex network of inter- and intrahelical salt bridges. Circular dichroism spectroscopy and analytical ultracentrifugation show that this peptide is highly α-helical and 100% dimeric under physiological buffer conditions. Interestingly, the peptide was shown to switch its oligomerization state from a dimer to a trimer upon increasing ionic strength. The correctness of the rational design principles used here is supported by details of the atomic structure of the peptide deduced from X-ray crystallography. The structure of the peptide shows that it is not a molten globule but assumes a unique, native-like conformation. This de novo peptide thus represents an attractive model system for the design of a molecular recognition system.

The yrdC family of genes codes for proteins that occur both independently and as a domain in proteins that have been implicated in regulation. An example for the latter case is the sua5 gene from yeast. Sua5 was identified as a suppressor of a translation initiation defect in cytochrome c and is required for normal growth in yeast (Na JG, Pinto I, Hampsey M, 1992, Genetics 11:791–801). However, the function of the Sua5 protein remains unknown; Sua5 could act either at the transcriptional or the posttranscriptional levels to compensate for an aberrant translation start codon in the cyc gene. To potentially learn more about the function of YrdC and proteins featuring this domain, the crystal structure of the YrdC protein from Escherichia coli was determined at a resolution of 2.0 Å. YrdC adopts a new fold with no obvious similarity to those of other proteins with known three-dimensional (3D) structure. The protein features a large concave surface on one side that exhibits a positive electrostatic potential. The dimensions of this depression, its curvature, and the fact that conserved basic amino acids are located at its floor suggest that YrdC may be a nucleic acid binding protein. An investigation of YrdC's binding affinities for single- and double-stranded RNA and DNA fragments as well as tRNAs demonstrates that YrdC binds preferentially to double-stranded RNA. Our work provides evidence that 3D structures of functionally uncharacterized gene products with unique sequences can yield novel folds and functional insights.

Previous conformational analysis of 10-residue linear peptides enabled us to identify some cross-strand side-chain interactions that stabilize β-hairpin conformations. The stabilizing influence of these interactions appeared to be greatly reduced when the interaction was located at the N- and C-termini of these 10-residue peptides. To investigate the effect of the position relative to the turn of favorable interactions on β-hairpin formation, we have designed two 15-residue β-hairpin forming peptides with the same residue composition and differing only in the location of two residues within the strand region. The conformational properties of these two peptides in aqueous solution were studied by 1H and 13C NMR. Differences in the conformational behavior of the two designed 15-residue peptides suggest that the influence of stabilizing factors for β-hairpin formation, in particular, cross-strand side-chain interactions, depends on their proximity to the turn. Residues adjacent to the turn are most efficient in that concern. This result agrees with the proposal that the turn region acts as the driving force in β-hairpin folding.

The SH3 domain, comprised of approximately 60 residues, is found within a wide variety of proteins, and is a mediator of protein–protein interactions. Due to the large number of SH3 domain sequences and structures in the databases, this domain provides one of the best available systems for the examination of sequence and structural conservation within a protein family. In this study, a large and diverse alignment of SH3 domain sequences was constructed, and the pattern of conservation within this alignment was compared to conserved structural features, as deduced from analysis of eighteen different SH3 domain structures. Seventeen SH3 domain structures solved in the presence of bound peptide were also examined to identify positions that are consistently most important in mediating the peptide-binding function of this domain. Although residues at the two most conserved positions in the alignment are directly involved in peptide binding, residues at most other conserved positions play structural roles, such as stabilizing turns or comprising the hydrophobic core. Surprisingly, several highly conserved side-chain to main-chain hydrogen bonds were observed in the functionally crucial RT-Src loop between residues with little direct involvement in peptide binding. These hydrogen bonds may be important for maintaining this region in the precise conformation necessary for specific peptide recognition. In addition, a previously unrecognized yet highly conserved β-bulge was identified in the second β-strand of the domain, which appears to provide a necessary kink in this strand, allowing it to hydrogen bond to both sheets comprising the fold.

Chaperonins cpn60/cpn10 (GroEL/GroES in Escherichia coli) assist folding of nonnative polypeptides. Folding of the chaperonins themselves is distinct in that it entails assembly of a sevenfold symmetrical structure. We have characterized denaturation and renaturation of the recombinant human chaperonin 10 (cpn10), which forms a heptamer. Denaturation induced by chemical denaturants urea and guanidine hydrochloride (GuHCl) as well as by heat was monitored by tyrosine fluorescence, far-ultraviolet circular dichroism, and cross-linking; all denaturation reactions were reversible. GuHCl-induced denaturation was found to be cpn10 concentration dependent, in accord with a native heptamer to denatured monomer transition. In contrast, urea-induced denaturation was not cpn10 concentration dependent, suggesting that under these conditions cpn10 heptamers denature without dissociation. There were no indications of equilibrium intermediates, such as folded monomers, in either denaturant. The different cpn10 denatured states observed in high [GuHCl] and high [urea] were supported by cross-linking experiments. Thermal denaturation revealed that monomer and heptamer reactions display the same enthalpy change (per monomer), whereas the entropy-increase is significantly larger for the heptamer. A thermodynamic cycle for oligomeric cpn10, combining chemical denaturation with the dissociation constant in absence of denaturant, shows that dissociated monomers are only marginally stable (3 kJ/mol). The thermodynamics for co-chaperonin stability appears conserved; therefore, instability of the monomer could be necessary to specify the native heptameric structure.

A challenge in computational protein folding is to assemble secondary structure elements—helices and strands—into well-packed tertiary structures. Particularly difficult is the formation of β-sheets from strands, because they involve large conformational searches at the same time as precise packing and hydrogen bonding. Here we describe a method, called Geocore-2, that (1) grows chains one monomer or secondary structure at a time, then (2) disconnects the loops and performs a fast rigid-body docking step to achieve canonical packings, then (3) in the case of intrasheet strand packing, adjusts the side-chain rotamers; and finally (4) reattaches loops. Computational efficiency is enhanced by using a branch-and-bound search in which pruning rules aim to achieve a hydrophobic core and satisfactory hydrogen bonding patterns. We show that the pruning rules reduce computational time by 103- to 105-fold, and that this strategy is computationally practical at least for molecules up to about 100 amino acids long.

Proteins often require cofactors to perform their biological functions and must fold in the presence of their cognate ligands. Using circular dichroism spectroscopy, we investigated the effects of divalent metal binding upon the folding pathway of Escherichia coli RNase HI. This enzyme binds divalent metal in its active site, which is proximal to the folding core of RNase HI as defined by hydrogen/deuterium exchange studies. Metal binding increases the apparent stability of native RNase HI chiefly by reducing the unfolding rate. As with the apo-form of the protein, refolding from high denaturant concentrations in the presence of Mg2+ follows three-state kinetics: formation of a rapid burst phase followed by measurable single exponential kinetics. Therefore, the overall folding pathway of RNase HI is minimally perturbed by the presence of metal ions. Our results indicate that the metal cofactor enters the active site pocket only after the enzyme reaches its native fold, and therefore, divalent metal binding stabilizes the protein by decreasing its unfolding rate. Furthermore, the binding of the cofactor is dependent upon a carboxylate critical for activity (Asp10). A mutation in this residue (D10A) alters the folding kinetics in the absence of metal ions such that they are similar to those observed for the unaltered enzyme in the presence of metal.