Ribonuclease T1: Structure,Function, and Stability

Proteins carry out the most important and difficult tasks in all living organisms. To do so, they must often interact specifically with other small and large molecules. This requires that they fold to a globular conformation with a unique active site that is used for the specific interaction. Consequently, protein folding can be regarded as the “secret of life”. Biochemists and chemists have a great interest in elucidating the mechanism by which proteins fold and in predicting the folded conformation and its stability given just the amino acid sequence. This challenge is sometimes called the “protein folding problem”. The ability to construct proteins differing in sequence by one or more amino acids and to analyze their three-dimensional structures by X-ray crystallography and NMR spectroscopy is a powerful tool for investigating the conformational stability and folding of proteins. Several proteins are now under intensive study by this approach. One of these is ribonuclease T1.     

Group II chaperonins as mediators of cytosolic protein folding

Protein folding and assembly in the cell requires the assistance of molecular chaperones. These components prevent off-pathway folding reactions that lead to aggregation. They are also critical factors in organismal stress physiology, protecting cells against heat shock and providing thermotolerance. Among this important protein family are chaperonins. They form large cylindrical double ring complexes with a central cavity where protein binding and folding takes place in an ATP-dependent manner. Recently, components functionally related to the eubacterial and organellar chaperonins have been found in the cytosol of archaebacteria and of eukaryotic cells. Based on their sequences and structural features, they have been classified as group II chaperonins, to distinguish them from the group I chaperonins occurring in bacteria. Of particular interest in the group II family is the eukaryotic CCT complex, whose function in protein folding and assembly has been demonstrated mainly for the cytoskeletal proteins tubulin and actin. Together with the Hsp70 chaperone system, it can be considered as an essential helper factor to facilitate the folding of native proteins in the eukaryotic cytosol. Recent structural data have opened the path to a molecular understanding of group II chaperonins and have helped to define their role in cellular protein folding.     

Chaperone-assisted protein folding in the cell cytoplasm

Folding of polypeptides in the cell typically requires the assistance of a set of proteins termed molecular chaperones. Chaperones are an essential group of proteins necessary for cell viability under both normal and stress conditions. There are several chaperone systems which carry out a multitude of functions all aimed towards insuring the proper folding of target proteins. Chaperones can assist in the efficient folding of newly-translated proteins as these proteins are being synthesized on the ribosome and can maintain pre-existing proteins in a stable conformation. Chaperones can also promote the disaggregation of preformed protein aggregates. Many of the identified chaperones are also heat shock proteins. The general mechanism by which chaperones carry out their function usually involves multiple rounds of regulated binding and release of an unstable conformer of target polypeptides. The four main chaperone systems in the Escherichia coli cytoplasm are as follows. (1) Ribosome-associated trigger factor that assists in the folding of newly-synthesized nascent chains. (2) The Hsp 70 system consisting of DnaK (Hsp 70), its cofactor DnaJ (Hsp 40), and the nucleotide exchange factor GrpE. This system recognizes polypeptide chains in an extended conformation. (3) The Hsp 60 system, consisting of GroEL (Hsp 60) and its cofactor GroES (Hsp 10), which assists in the folding of compact folding intermediates that expose hydrophobic surfaces. (4) The Clp ATPases which are typically members of the Hsp 100 family of heat shock proteins. These ATPases can unfold proteins and disaggregate preformed protein aggregates to target them for degradation. Several advances have recently been made in characterizing the structure and function of all of these chaperone systems. These advances have provided us with a better understanding of the protein folding process in the cell.     

Molecular simulation of protein dynamics in nanopores. I. Stability and folding

Discontinuous molecular dynamics simulations, together with the protein intermediate resolution model, an intermediate-resolution model of proteins, are used to carry out several microsecond-long simulations and study folding transition and stability of alpha-de novo-designed proteins in slit nanopores. Both attractive and repulsive interaction potentials between the proteins and the pore walls are considered. Near the folding temperature T(f) and in the presence of the attractive potential, the proteins undergo a repeating sequence of folding/partially folding/unfolding transitions, with T(f) decreasing with decreasing pore sizes. The unfolded states may even be completely adsorbed on the pore's walls with a negative potential energy. In such pores the energetic effects dominate the entropic effects. As a result, the unfolded state is stabilized, with a folding temperature T(f) which is lower than its value in the bulk and that, compared with the bulk, the folding rate decreases. The opposite is true in the presence of a repulsive interaction potential between the proteins and the walls. Moreover, for short proteins in very tight pores with attractive walls, there exists an unfolded state with only one alpha-helical hydrogen bond and an energy nearly equal to that of the folded state. The proteins have, however, high entropies, implying that they cannot fold onto their native structure, whereas in the presence of repulsive walls the proteins do attain their native structure. There is a pronounced asymmetry between the two termini of the protein with respect to their interaction with the pore walls. The effect of a variety of factors, including the pore size and the proteins' length, as well as the temperature, is studied in detail.     

Highly ordered structures of peptides by using molecular scaffolds

Protein secondary structures such as alpha-helices, beta-sheets, and beta-turns are important in inducing the three-dimensional structure and biological activity of proteins. Designing secondary structure mimics composed of short peptides has attracted much attention not only to gain fundamental insight into the factors affecting protein folding but also to develop pharmacologically useful compounds, artificial receptors, asymmetric catalysts, and new materials. In this tutorial review, we focus on molecular scaffolds employed to induce beta-sheet-like structure in attached peptide chains, thereby creating highly ordered molecular structures, and discuss the versatility of these molecular scaffolds to regulate the attached peptide strands in the appropriate dimensions.     

Enzymes catalyzing protein folding and their cellular functions

In live cells, protein folding often cannot occur spontaneously, but requires the participation of helper proteins - molecular chaperones and foldases. The mechanisms employed by chaperones markedly increase the effectiveness of protein folding, but have no bearing on the rate of this process, whereas foldases actually accelerate protein folding by exerting a direct influence on the rate-limiting steps of the overall reaction. Two types of foldases are known, using different principles of action. Peptidyl-prolyl cis/trans isomerase and protein-disulfide isomerase catalyze the folding of every protein that needs isomerization of prolyl peptide bonds or formation and isomerization of disulfide bonds for proper folding. By contrast, some foldases operating in the periplasm of bacterial cells are specifically designed to help in the folding of substrate proteins whose primary structure does not contain sufficient information for correct folding. In this review, we discuss recent data on the catalytic mechanisms of both types of foldases, focusing specifically on how a catalyst provides the structural information required for the folding of a target protein. Comparative analysis of the mechanisms employed by two different periplasmic foldases is used to substantiate the notion that combinations of a protein which is unable to fold independently and a specific catalyst delivering the necessary steric information are probably designed to achieve some particular biological purposes. The review also covers the problem of participation of peptidyl-prolyl cis/trans isomerase in different cellular functions, highlighting the role of this enzyme in conformational rearrangements of folded native proteins.     

微流控超快混合器及生物大分子折叠动力学应用研究进展

生物大分子如蛋白质或核酸的功能与其三维结构密切相关,折叠动力学研究可揭示生物大分子从自由的一级结构形成具有活性高级结构的动态过程,近年来倍受科学界重视。生物大分子的折叠过程一般发生在毫秒、微秒甚至是亚微秒时间水平,而启动折叠反应则需在更短的时间内完成。基于微流控芯片的超快混合器能使溶液在短时间内达到完全混合从而触发反应,已被广泛应用于生物大分子折叠动力学研究。该文系统评述了超快微混合器的国内外研究进展,并介绍了其在大分子折叠动力学研究中的应用。     

Flexible Folding: Disulfide-Containing Peptides and Proteins Choose the Pathway Depending on the Environments

In the last few decades, development of novel experimental techniques, such as new types of disulfide (SS)-forming reagents and genetic and chemical technologies for synthesizing designed artificial proteins, is opening a new realm of the oxidative folding study where peptides and proteins can be folded under physiologically more relevant conditions. In this review, after a brief overview of the historical and physicochemical background of oxidative protein folding study, recently revealed folding pathways of several representative peptides and proteins are summarized, including those having two, three, or four SS bonds in the native state, as well as those with odd Cys residues or consisting of two peptide chains. Comparison of the updated pathways with those reported in the early years has revealed the flexible nature of the protein folding pathways. The significantly different pathways characterized for hen-egg white lysozyme and bovine milk α-lactalbumin, which belong to the same protein superfamily, suggest that the information of protein folding pathways, not only the native folded structure, is encoded in the amino acid sequence. The application of the flexible pathways of peptides and proteins to the engineering of folded three-dimensional structures is an interesting and important issue in the new realm of the current oxidative protein folding study.     

Robustness of atomistic Gō models in predicting native-like folding intermediates

Go? models are exceedingly popular tools in computer simulations of protein folding. These models are native-centric, i.e., they are directly constructed from the protein's native structure. Therefore, it is important to understand up to which extent the atomistic details of the native structure dictate the folding behavior exhibited by Go? models. Here we address this challenge by performing exhaustive discrete molecular dynamics simulations of a Go? potential combined with a full atomistic protein representation. In particular, we investigate the robustness of this particular type of Go? models in predicting the existence of intermediate states in protein folding. We focus on the N47G mutational form of the Spc-SH3 folding domain (x-ray structure) and compare its folding pathway with that of alternative native structures produced in silico. Our methodological strategy comprises equilibrium folding simulations, structural clustering, and principal component analysis.     

Molecular Machines: How Motion and Other Functions of Living Organisms Can Result from Reversible Chemical Changes

Certain model proteins dramatically fold and become more ordered on raising the temperature. When the temperature is raised to drive folding and assembly, these model proteins can lift weights and perform work; they can produce motion. The temperature of warm-blooded animals, however, is kept constant. Therefore, motion cannot result from a change in temperature. In this case, a free energy change, caused, for example, by an increase in the concentration of a chemical, can lower the temperature at which the protein folding and assembly transition occurs from above to below physiological temperature. Raising the concentration of a chemical isothermally has indeed been shown to result in motion and the efficient performance of work. These model proteins and the mechanism they reveal provide insight into the molecular basis for diverse biological functions; they are models for the molecular machines that comprise the living organism, and they provide a new class of materials for both medical and nonmedical applications.     

液相色谱中一个新的表征参数Z Ⅳ.反相液相色谱中甲酸浓度与生物大分子的构象变化

反相高效液相色谱中蛋白质分子的Z值可以作为平衡状态下蛋白分子构象变化的表征,它与蛋白分子起始状态构象无关。在异丙醇-甲酸-水体系中蛋白的Z值会因流动相中甲酸浓度的增大而减小。除蛋白分子构象变化因素外,蛋白Z值的变化也与甲酸参与蛋白与置换剂分子间的计量置换过程有关。在蛋白分子未完全失去分子立体结构时,其Z值与分子量的立方根成正比。当甲酸浓度足够大时,例如大于40％(V/V)时,蛋白分子完全失去三维结构。     

Influence of the native topology on the folding barrier for small proteins

The possibility of downhill instead of two-state folding for proteins has been a very controversial topic which arose from recent experimental studies. From the theoretical side, this question has also been accomplished in different ways. Given the experimental observation that a relationship exists between the native structure topology of a protein and the kinetic and thermodynamic properties of its folding process, Gō-type potentials are an appropriate way to approach this problem. In this work, we employ an interaction potential from this family to get a better insight on the topological characteristics of the native state that may somehow determine the presence of a thermodynamic barrier in the folding pathway. The results presented here show that, indeed, the native topology of a small protein has a great influence on its folding behavior, mostly depending on the proportion of local and long range contacts the protein has in its native structure. Furthermore, when all the interactions present contribute in a balanced way, the transition results to be cooperative. Otherwise, the tendency to a downhill folding behavior increases.     

Foldable subunits of helix protein

Detection of foldable subunits in proteins is an important approach to understand their evolutions and find building motifs for de novo protein design. Using united-residue model, we simulated the folding of a six-helix protein with a length of 120 amino acids (C-terminal domain of Ku86). The folding behaviors, structural topology and sequence repetition of this protein all suggest that it may have a two-fold quasi-repetition or symmetry in its sequence and structure. Therefore, we simulated the folding of its two halves (1–60 and 61–120 amino acids) and find that they can fold into native conformations independently. It is also found that their folding behaviors are very similar to other three-helix bundles. This suggests that this protein may be divided into two foldable halves.     

Topology-based models and NMR structures in protein folding simulations

Topology-based interaction potentials are simplified models that use the native contacts in the folded structure of a protein to define an energetically unfrustrated folding funnel. They have been widely used to analyze the folding transition and pathways of different proteins through computer simulations. Obviously, they need a reliable, experimentally determined folded structure to define the model interactions. In structures elucidated through NMR spectroscopy, a complex treatment of the raw experimental data usually provides a series of models, a set of different conformations compatible with the available experimental data. Here, we use an efficient coarse-grained simulation technique to independently consider the contact maps from every different NMR model in a protein whose structure has been resolved by the use of NMR spectroscopy. For lambda-Cro repressor, a homodimeric protein, we have analyzed its folding characteristics with a topology-based model. We have focused on the competition between the folding of the individual chains and their binding to form the final quaternary structure. From 20 different NMR models, we find a predominant three-state folding behavior, in agreement with experimental data on the folding pathway for this protein. Individual NMR models, however, show distinct characteristics, which are analyzed both at the level of the interplay between tertiary/quaternary structure formation and also regarding the thermal stability of the tertiary structure of every individual chain.     

Estimation of protein folding rate from Monte Carlo simulations and entropy capacity

The problem of protein self-organization is one of the most important problems of molecular biology nowadays. Despite the recent success in the understanding of general principles of protein folding, details of this process are yet to be elucidated. Moreover, the prediction of protein folding rates has its own practical value due to the fact that aggregation directly depends on the rate of protein folding. The time of folding has been calculated for 67 proteins with known experimental data at the point of thermodynamic equilibrium between unfolded and native states using a Monte Carlo model where each residue is considered to be either folded as in the native state or completely disordered. The times of folding for 67 proteins which reach the native state within the limit of 10(8) Monte Carlo steps are in a good correlation with the experimentally measured folding rate at the mid-transition point (the correlation coefficient is -0.82). Theoretical consideration of a capillarity model for the process of protein folding demonstrates that the difference in the folding rate for proteins sharing more spherical and less spherical folds is the result of differences in the conformational entropy due to a larger surface of the boundary between folded and unfolded phases in the transition state for proteins with more spherical fold. The capillarity model allows us to predict the folding rate at the same level of correlation as by Monte Carlo simulations. The calculated model entropy capacity (conformational entropy per residue divided by the average contact energy per residue) for 67 proteins correlates by about 78% with the experimentally measured folding rate at the mid-transition point.     

Folding of elongated proteins: conventional or anomalous?

The complexity of the mechanisms by which proteins fold has been shown by many studies to be governed by their native-state topologies. This was manifested in the ability of the native topology-based model to capture folding mechanisms and the success of folding rate predictions based on various topological measures, such as the contact order. However, while the finer details of topological complexity have been thoroughly examined and related to folding kinetics, simpler characteristics of the protein, such as its overall shape, have been largely disregarded. In this study, we investigated the folding of proteins with an unusual elongated geometry that differs substantially from the common globular structure. To study the effect of the elongation degree on the folding kinetics, we used repeat proteins, which become more elongated as they include more repeating units. Some of these have apparently anomalous experimental folding kinetics, with rates that are often less than expected on the basis of rates for globular proteins possessing similar topological complexity. Using experimental folding rates and a larger set of rates obtained from simulations, we have shown that as the protein becomes increasingly elongated, its folding kinetics becomes slower and deviates more from the rate expected on the basis of topology measures fitted for globular proteins. The observed slow kinetics is a result of a more complex pathway in which stable intermediates composed of several consecutive repeats can appear. We thus propose a novel measure, an elongation-sensitive contact order, that takes into account both the extent of elongation and the topological complexity of the protein. This new measure resolves the apparent discrimination between the folding of globular and elongated repeat proteins. Our study extends the current capabilities of folding-rate predictions by unifying the kinetics of repeat and globular proteins.     

A conventional an 2DCOS infrared approach to the kinetics of protein misfolding

Cell viability depends on the correct folding of the proteins involved in metabolism. Proteins are synthesized on the endoplasmic reticulum and must follow a pathway to a correct, metastable, tridimensional structure. Changes in structure or in environmental conditions can drive an instability of the folding conditions and produce non-active aggregates that in principle are proteolysed by the cellular mechanisms. However, these aggregates can be even more stable than the native proteins, escaping the cellular control. They can be classified as amorphous, if there is not a well-organized structural pattern, or ordered if a repetitive pattern is produced. These ordered structures, known as fibrils, are involved in many diseases. Infrared spectroscopy is a method of choice to study its formation because it is not affected by turbidity or the formation of high molecular weight aggregates. Moreover, in both cases, two bands characteristic of intermolecular β-sheets allow the monitoring of the aggregate formation. In both cases, the appearance of these bands involves a non-reversible path in protein folding. It has been suggested that a difference in the ordered structures involves an increasing in band intensity. This change can be the origin in variations on the 2DCOS maps. The synchronous map gives an overall idea of the process involved. The asynchronous is more informative because reflects the kinetic changes produced. The outcome of both processes, amorphous or ordered is that 2DCOS can provide a further insight to the knowledge of the kinetic processes giving rise to aggregated structures. This outcome could consist on the order in which the different secondary structures are prone to form the aggregates.     

全新设计蛋白质的折叠机制

蛋白质全新设计和折叠研究是从两个不同的方向来理解蛋白质序列-结构-功能关系这一结构生物学重要问题. 蛋白质全新设计取得的成功实例一定程度上检验了人们对蛋白质结构和相互作用理解的准确性, 但它们中多数所表现的不同于天然蛋白质的折叠动力学特征也表明, 要达到最终的功能化实现目标还面临着不少的挑战. 本文综述了蛋白质全新设计的发展过程及现状, 蛋白质折叠研究在实验、理论及模拟方面的研究进展, 以及全新设计蛋白质的折叠机制的研究现状. 阐述了深入了解全新设计蛋白质与天然蛋白质折叠机制的不同, 可以为进一步有效地合理化设计蛋白质提供有益的参考.