Protein folding: construction of functional structure

The problem of protein folding was studied with trypsin inhibitor by deviation analysis (1). The results showed that: i) Qualitatively, the main features of the structure, determined by this method, coincided with the structure determined by X-ray crystallography (3). This structure is, however, not topological but functional, and may elucidate the functional relations between various parts of the protein.     

Protein folding: from the levinthal paradox to structure prediction.

This article is a personal perspective on the developments in the field of protein folding over approximately the last 40 years. In addition to its historical aspects, the article presents a view of the principles of protein folding with particular emphasis on the relationship of these principles to the problem of protein structure prediction. It is argued that despite much that is new, the essential elements of our current understanding of protein folding were anticipated by researchers many years ago. These elements include the recognition of the central importance of the polypeptide backbone as a determinant of protein conformation, hierarchical protein folding, and multiple folding pathways. Important areas of progress include a detailed characterization of the folding pathways of a number of proteins and a fundamental understanding of the physical chemical forces that determine protein stability. Despite these developments, fold prediction algorithms still encounter difficulties in identifying the correct fold for a given sequence. This may be due to the possibility that the free energy differences between at least a few alternate conformations of many proteins are not large. Significant progress in protein structure prediction has been due primarily to the explosive growth of sequence and structural databases. However, further progress is likely to depend in part on the ability to combine information available from databases with principles and algorithms derived from physical chemical studies of protein folding. An approach to the integration of the two areas is outlined with specific reference to the PrISM program that is a fully integrated sequence/structural-analysis/fold-recognition/homology model building software system.     

Protein folding mechanisms: new methods and emerging ideas

During the past year, advances in our understanding of folding mechanisms have been made through detailed experimental and theoretical studies of a number of proteins. The development of new methods has allowed the earliest events in folding to be probed and the measurement of folding at the level of individual molecules is now possible, opening the door to exciting new experiments.     

Protein folding and unfolding simulations: a new challenge for data mining

One of the unsolved paradigms in molecular biology is the protein folding problem. In recent years, with the identification of several diseases as protein folding disorders and with the explosion of genome information and the need for efficient ways to predict protein structure, protein folding became a central issue in molecular sciences research. Using molecular dynamics unfolding simulations of an amyloidogenic protein--transthyretin--as an example, we put forward a series of ideas on how simulations of this type may be used to infer rules and unfolding behavior in amyloidogenic proteins, and to extrapolate rules for protein folding in different structural classes of proteins. These, in turn, could help in the development of protein structure prediction methods. The need to analyse different proteins and to run multiple simulations creates a huge amount of data which has to be stored, managed, analyzed and shared (database and Grid technology; data mining). Once the data is captured, the next challenge is to find meaningful patterns (associations, correlations, clusters, rules, relationships) among molecular properties, or their relative importance at different stages of the folding or unfolding processes. This clearly puts new and interesting challenges to the bioinformatics community.     

Protein structure prediction using a docking-based hierarchical folding scheme

The pathways by which proteins fold into their specific native structure are still an unsolved mystery. Currently, many methods for protein structure prediction are available, and most of them tackle the problem by relying on the vast amounts of data collected from known protein structures. These methods are often not concerned with the route the protein follows to reach its final fold. This work is based on the premise that proteins fold in a hierarchical manner. We present FOBIA, an automated method for predicting a protein structure. FOBIA consists of two main stages: the first finds matches between parts of the target sequence and independently folding structural units using profile-profile comparison. The second assembles these units into a 3D structure by searching and ranking their possible orientations toward each other using a docking-based approach. We have previously reported an application of an initial version of this strategy to homology based targets. Since then we have considerably enhanced our method's abilities to allow it to address the more difficult template-based target category. This allows us to now apply FOBIA to the template-based targets of CASP8 and to show that it is both very efficient and promising. Our method can provide an alternative for template-based structure prediction, and in particular, the docking-basedranking technique presented here can be incorporated into any profile-profile comparison based method.     

Protein disulfide isomerase: the structure of oxidative folding

Cellular functions hinge on the ability of proteins to adopt their correct folds, and misfolded proteins can lead to disease. Here, we focus on the proteins that catalyze disulfide bond formation, a step in the oxidative folding pathway that takes place in specialized cellular compartments. In the endoplasmic reticulum of eukaryotes, disulfide formation is catalyzed by protein disulfide isomerase (PDI); by contrast, prokaryotes produce a family of disulfide bond (Dsb) proteins, which together achieve an equivalent outcome in the bacterial periplasm. The recent crystal structure of yeast PDI has increased our understanding of the function and mechanism of PDI. Comparison of the structure of yeast PDI with those of bacterial DsbC and DsbG reveals some similarities but also striking differences that suggest directions for future research aimed at unraveling the catalytic mechanism of disulfide bond formation in the cell.     

Protein folding as a diffusional process.

A protein chain must move relative to the solvent molecules and explore many conformations when it folds from the extended unfolded state to the compact native state. Experimental and theoretical approaches suggest that diffusional processes in fact contribute to the kinetics of protein folding. We describe here how variations of the solvent viscosity can be employed to uncover the diffusional contributions to a folding reaction and assess the use of transition state theory and Kramers' rate theory for the analysis of protein folding reactions.     

Protein folding: matching theory and experiment.

The impact of folding funnels and folding simulations on the way experimentalists interpret results is examined. The image of the transition state has changed from a unique species that has a strained configuration, with a correspondingly high free energy, to a more ordinary folding intermediate, whose balance between limited conformational entropy and stabilizing contacts places it at the top of the free energy barrier. Evidence for a broad transition barrier comes from studies showing that mutations can change the position of the barrier. The main controversial issue now is whether populated folding intermediates are productive on-pathway intermediates or dead-end traps. Direct experimental evidence is needed. Theories suggesting that populated intermediates are trapped in a glasslike state are usually based on mechanisms which imply that trapping would only be extremely short-lived (e.g., nanoseconds) in water at 25 degrees C. There seems to be little experimental evidence for long-lived trapping in monomers, if folding aggregates are excluded. On the other hand, there is good evidence for kinetic trapping in dimers. alpha-Helix formation is currently the fastest known process in protein folding, and incipient helices are present at the start of folding. Fast helix formation has the effect of narrowing drastically the choice of folding routes. Thus helix formation can direct folding. It changes the folding metaphor from pouring liquid down a folding funnel to a train leaving a switchyard with only a few choices of exit tracks.     

Protein folding and maturation in a cell-free system.

Reduced cellular systems have provided important tools to study complex cellular processes. Here we describe the oxidation, oligomerization, and chaperone binding of the viral glycoprotein influenza hemagglutinin in a cell-free system. The cell-free system, comprised of rough endoplasmic reticulum derived microsomes and a reticulocyte lysate, supported the complete maturation of hemagglutinin from the earliest oxidative intermediate to the mature homo-oligomer. Hemagglutinin disulfide bond formation and oligomerization were found to occur in a time- and temperature-dependent manner. Hemagglutinin's temporal association with the molecular chaperones calnexin and calreticulin was similar to that observed for their association with elongating ribosome-attached nascent chains in live cells. Furthermore, a procedure is described that permits the translocation of protein into microsomes that are depleted of lumenal contents. This cell-free system, therefore, provided an effective means to study the biological maturation processes of a protein that traverses the secretory pathway.     

Protein folding and protein refolding.

The functional three-dimensional structure of proteins is determined solely by their amino acid sequences. Protein folding occurs spontaneously beginning with the formation of local secondary structure concomitant with a compaction of the molecule. Secondary structure elements subsequently interact to form subdomains and domains stabilized by tertiary interactions. Disulfide bond formation, and cis-trans isomerization of X-Pro peptide bonds, as the rate-limiting folding reactions, are enzymatically catalyzed during protein folding in the cell. Although folding of domains is fast enough to occur cotranslationally in vivo, such vectorial folding on the ribosome is not essential for attainment of the native structure of a protein. Slow steps on the pathway to the functional protein structure are docking reactions of domains, association of subunits, or reshuffling reactions at the oligomer level. Aggregation as a competing side reaction is prevented, and the kinetic partition between competing polypeptide folding and translocation reactions is regulated by chaperone proteins binding to incompletely folded polypeptides.     

Protein folding in vitro.

It is becoming increasingly evident that intermediates observed in protein folding in vitro may be closely related to conformational states that are important in various intracellular processes. This review focuses on recent advances in in vitro protein-folding studies with particular reference to the molten globule state, which is purported to be a common and distinct intermediate of protein folding.     

Oxidative stress: Protein folding with a novel redox switch.

A novel cellular response to oxidative stress has been discovered, in which the activity of a molecular chaperone, Hsp33, is modulated by the environmental redox potential. This provides a rapid first defence mechanism against the potentially very harmful toxic effects of oxidative stress.     

Protein folding in a specialized compartment: the endoplasmic reticulum.

The endoplasmic reticulum ensures proper folding of secretory proteins. In this review, we summarize and discuss the functions of different classes of folding mediators in the secretory pathway and propose updated models of the quality control system.