The structural basis of protein folding and its links with human disease

The ability of proteins to fold to their functional states following synthesis in the intracellular environment is one of the most remarkable features of biology. Substantial progress has recently been made towards understanding the fundamental nature of the mechanism of the folding process. This understanding has been achieved through the development and concerted application of a variety of novel experimental and theoretical approaches to this complex problem. The emerging view of folding is that it is a stochastic process, but one biased by the fact that native-like interactions between residues are on average more stable than non-native ones. The sequences of natural proteins have emerged through evolutionary processes such that their unique native states can be found very efficiently even in the complex environment inside a living cell. But under some conditions proteins fail to fold correctly, or to remain correctly folded, in living systems, and this failure can result in a wide range of diseases. One group of diseases, known as amyloidoses, which includes Alzheimer's and the transmissible spongiform encephalopathies, involves deposition of aggregated proteins in a variety of tissues. These diseases are particularly intriguing because evidence is accumulating that the formation of the highly organized amyloid aggregates is a generic property of polypeptides, and not simply a feature of the few proteins associated with recognized pathological conditions. That such aggregates are not normally found in properly functional biological systems is again a testament to evolution, in this case of a variety of mechanisms inhibiting their formation. Understanding the nature of such protective mechanisms is a crucial step in the development of strategies to prevent and treat these debilitating diseases.     

Inversion of the balance between hydrophobic and hydrogen bonding interactions in protein folding and aggregation

Identifying the forces that drive proteins to misfold and aggregate, rather than to fold into their functional states, is fundamental to our understanding of living systems and to our ability to combat protein deposition disorders such as Alzheimer's disease and the spongiform encephalopathies. We report here the finding that the balance between hydrophobic and hydrogen bonding interactions is different for proteins in the processes of folding to their native states and misfolding to the alternative amyloid structures. We find that the minima of the protein free energy landscape for folding and misfolding tend to be respectively dominated by hydrophobic and by hydrogen bonding interactions. These results characterise the nature of the interactions that determine the competition between folding and misfolding of proteins by revealing that the stability of native proteins is primarily determined by hydrophobic interactions between side-chains, while the stability of amyloid fibrils depends more on backbone intermolecular hydrogen bonding interactions.     

Helix formation and stability in membranes

In this article we review current understanding of basic principles for the folding of membrane proteins, focusing on the more abundant alpha-helical class. Membrane proteins, vital to many biological functions and implicated in numerous diseases, fold into their active conformations in the complex environment of the cell bilayer membrane. While many membrane proteins rely on the translocon and chaperone proteins to fold correctly, others can achieve their functional form in the absence of any translation apparatus or other aides. Nevertheless, the spontaneous folding process is not well understood at the molecular level. Recent findings suggest that helix fraying and loop formation may be important for overall structure, dynamics and regulation of function. Several types of membrane helices with ionizable amino acids change their topology with pH. Additionally we note that some peptides, including many that are rich in arginine, and a particular analogue of gramicidin, are able passively to translocate across cell membranes. The findings indicate that a final protein structure in a lipid-bilayer membrane is sequence-based, with lipids contributing to stability and regulation. While much progress has been made toward understanding the folding process for alpha-helical membrane proteins, it remains a work in progress. This article is part of a Special Issue entitled: Emergence of Complex Behavior in Biomembranes edited by Marjorie Longo.     

Insights into chaperonin function from studies on archaeal thermosomes

It is now well understood that, although proteins fold spontaneously (in a thermodynamic sense), many nevertheless require the assistance of helpers called molecular chaperones to reach their correct and active folded state in living cells. This is because the pathways of protein folding are full of traps for the unwary: the forces that drive proteins into their folded states can also drive them into insoluble aggregates, and, particularly when cells are stressed, this can lead, without prevention or correction, to cell death. The chaperonins are a family of molecular chaperones, practically ubiquitous in all living organisms, which possess a remarkable structure and mechanism of action. They act as nanoboxes in which proteins can fold, isolated from their environment and from other partners with which they might, with potentially deleterious consequences, interact. The opening and closing of these boxes is timed by the binding and hydrolysis of ATP. The chaperonins which are found in bacteria are extremely well characterized, and, although those found in archaea (also known as thermosomes) and eukaryotes have received less attention, our understanding of these proteins is constantly improving. This short review will summarize what we know about chaperonin function in the cell from studies on the archaeal chaperonins, and show how recent work is improving our understanding of this essential class of molecular chaperones.     

New insights into the mechanisms of protein misfolding and aggregation in amyloidogenic diseases derived from pressure studies

Hydrostatic pressure is a robust tool for studying the thermodynamics of protein folding and protein interactions, as well as the dynamics and structure of folding intermediates. One of the main innovations obtained from using high pressure is the stabilization of folding intermediates such as molten-globule conformations, thus providing a unique opportunity for characterizing their structure and dynamics. Equally important is the prospect of understanding protein misfolding diseases by using pressure to populate partially folded intermediates at the junction between productive and off-pathway folding, which may give rise to misfolded proteins, aggregates, and amyloids. High hydrostatic pressure (HHP) has also been used to dissociate nonamyloid aggregates and inclusion bodies. In many proteins, the competition between correct folding and misfolding can lead to formation of insoluble aggregates, an important problem for the biotechnology industry and for human pathologies such as amyloidosis, Alzheimer's, Parkinson's, prion's, and tumor diseases. The diversity of diseases that result from protein misfolding has made this theme an important research focus for pharmaceutical and biotechnology companies. The use of high-pressure promises to contribute to the identification of the mechanisms behind these defects and creation of therapies against these diseases.     

Understanding protein folding from globular to amyloid state: Aggregation: Darker side of protein

Folding and unfolding are crucial ways of modulating biological activity and targeting proteins to different cellular locations. In the living system, protein folding occurs in a very crowded environment, often assisted with helper proteins. In some cases this pathway can go off beam and the protein can either misfold or aggregate or form structures of elongated-unbranched morphology known as amyloid fibrils. Protein folding is not just an academic matter. Recombinant biotechnology and pharmaceutical industries are some of the fields where both theoretical and practical knowledge of protein folding is required. Misfolded protein and amyloid fibrils that escape the cellular quality control check are the basic reason of a number of increasingly widespread neurodegenerative diseases such as Alzheimer's and variant Creutzfeldt-Jakob etc. Thus, protein folding study also emerges as an interesting area in the field of biomedical research. This review deals with basic concepts related to protein folding and misfolding forming toxic aggregates and amyloid fibrils as well as disease associated with them. A more practical approach will be revealed to the early diagnosis of aggregation-prone diseases and amyloid states and their balanced therapeutics.     

Post-translocational folding of secretory proteins in Gram-positive bacteria

The transport of proteins from their site of synthesis in the cytoplasm to their functional location is an essential characteristic of all living cells. In Gram-positive bacteria the majority of proteins that are translocated across the cytoplasmic membrane are delivered to the membrane-cell wall interface in an essentially unfolded form. They must then be folded into their native configuration in an environment that is dominated by a high density of immobilised negative charge-in essence an ion exchange resin. It is essential to the viability of the cell that these proteins do not block the translocation machinery in the membrane, form illegitimate interactions with the cell wall or, through intermolecular interactions, form insoluble aggregates. Native Gram-positive proteins therefore have intrinsic folding characteristics that facilitate their rapid folding, and this is assisted by a variety of folding factors, including enzymes, peptides and metal ions. Despite these intrinsic and extrinsic factors, secretory proteins do misfold, particularly if the cell is subjected to certain types of stress. Consequently, Gram-positive bacteria such as Bacillus subtilis encode membrane- and cell wall-associated proteases that act as a quality control machine, clearing misfolded or otherwise aberrant proteins from the translocase and the cell wall.     

Pharmacologic rescue of conformationally-defective proteins: implications for the treatment of human disease

The process of quality control in the endoplasmic reticulum involves a variety of mechanisms which ensure that only correctly folded proteins enter the secretory pathway. Among these are conformation-screening mechanisms performed by molecular chaperones that assist in protein folding and prevent non-native (or misfolded) proteins from interacting with other misfolded proteins. Chaperones play a central role in the triage of newly formed proteins prior to their entry into the secretion, retention, and degradation pathways. Despite this stringent quality control mechanism, gain- or loss-of-function mutations that affect protein folding in the endoplasmic reticulum can manifest themselves as profound effects on the health of an organism. Understanding the molecular, cellular, and energetic mechanisms of protein routing could prevent or correct the structural abnormalities associated with disease-causing misfolded proteins. Rescue of misfolded, "trafficking-defective", but otherwise functional, proteins is achieved by a variety of physical, chemical, genetic, and pharmacological approaches. Pharmacologic chaperones (or "pharmacoperones") are template molecules that may potentially arrest or reverse diseases by inducing mutant proteins to adopt native-type-like conformations instead of improperly folded ones. Such restructuring leads to a normal pattern of cellular localization and function. This review focuses on protein misfolding and misrouting related to various disease states and describes promising approaches to overcoming such defects. Special attention is paid to the gonadotropin-releasing hormone receptor, since there is a great deal of information about this receptor, which has recently emerged as a particularly instructive model.     

Physicochemical bases for protein folding,dynamics, and protein-ligand binding

Proteins are essential parts of living organisms and participate in virtually every process within cells.As the genomic sequences for increasing number of organisms are completed,research into how proteins can perform such a variety of functions has become much more intensive because the value of the genomic sequences relies on the accuracy of understanding the encoded gene products.Although the static three-dimensional structures of many proteins are known,the functions of proteins are ultimately governed by their dynamic characteristics,including the folding process,conformational fluctuations,molecular motions,and protein-ligand interactions.In this review,the physicochemical principles underlying these dynamic processes are discussed in depth based on the free energy landscape(FEL)theory.Questions of why and how proteins fold into their native conformational states,why proteins are inherently dynamic,and how their dynamic personalities govern protein functions are answered.This paper will contribute to the understanding of structure-function relationship of proteins in the post-genome era of life science research.     

Outer membrane protein folding from an energy landscape perspective

The cell envelope is essential for the survival of Gram-negative bacteria. This specialised membrane is densely packed with outer membrane proteins (OMPs), which perform a variety of functions. How OMPs fold into this crowded environment remains an open question. Here, we review current knowledge about OMP folding mechanisms in vitro and discuss how the need to fold to a stable native state has shaped their folding energy landscapes. We also highlight the role of chaperones and the β-barrel assembly machinery (BAM) in assisting OMP folding in vivo and discuss proposed mechanisms by which this fascinating machinery may catalyse OMP folding.     

The trials and tribulations of membrane protein folding in vitro

Membrane proteins are hard to handle and consequently the purification of functional protein in milligram quantities is a major problem. One reason for this is that once integral membrane proteins are outside their native membrane, they are prone to aggregation, are unstable and are frequently only partially functional. Knowledge of membrane protein folding mechanisms in vitro can help to understand the causes of these problems and work toward strategies to disaggregate and fold proteins correctly. Kinetic and stability studies are emerging on membrane protein folding, mainly on bacterial proteins. Mutagenesis methods have also been used to probe specific structural features or bonds in proteins. In addition, manipulation of lipid properties can be used to improve the efficiency of folding as well as the stability and function of the protein.     

The molecular chaperone concept

Molecular chaperones are a ubiquitous family of cellular proteins which mediate the correct folding of other polypeptides, and in some cases their assembly into oligomeric structures, but which are not components of those final structures. Known chaperones do not possess steric information for protein folding but inhibit unproductive folding and assembly pathways which would otherwise act as dead-end kinetic traps and produce incorrect structures. Chaperones function by binding specifically and non-covalently to interactive protein surfaces that are exposed transiently during cellular processes such as protein synthesis, protein transport across membranes, DNA synthesis, the recycling of clathrin cages, the assembly of organellar complexes from imported subunits, and stress responses. This binding is reversed under circumstances which favour correct interactions and in some cases ATP hydrolysis is involved in this reversal. Some chaperones bind specifically to a structural feature present in a wide range of unrelated proteins that is accessible only during the early stages of folding. The nature of this structural feature is unknown, but its identification is an important goal of current research. Knowledge of chaperone function may be important for the production of proteins for biotechnological purposes since in some cases chaperones may improve the yield of functional product. It is likely that chaperone diseases exist which result from the failure of certain proteins to fold correctly due to changes in chaperone structure.     

The trials and tribulations of membrane protein folding in vitro

Membrane proteins are hard to handle and consequently the purification of functional protein in milligram quantities is a major problem. One reason for this is that once integral membrane proteins are outside their native membrane, they are prone to aggregation, are unstable and are frequently only partially functional. Knowledge of membrane protein folding mechanisms in vitro can help to understand the causes of these problems and work toward strategies to disaggregate and fold proteins correctly. Kinetic and stability studies are emerging on membrane protein folding, mainly on bacterial proteins. Mutagenesis methods have also been used to probe specific structural features or bonds in proteins. In addition, manipulation of lipid properties can be used to improve the efficiency of folding as well as the stability and function of the protein.     

药物分子伴侣: 蛋白质折叠运输缺陷的新疗法

大量遗传性疾病的发生是由于基因突变引起蛋白质错误折叠而不能运输到作用位点,从而导致功能缺陷．近年来兴起的药物分子伴侣是恢复蛋白质折叠运输缺陷的新疗法,这类化合物一般为目的蛋白的底物类似物、受体配基或酶抑制剂等化学小分子,具细胞通透性,能在内质网中特异性识别并结合突变蛋白,校正并稳定其正确构象,协助其运输到正确位点,直接恢复突变蛋白功能,可治疗各种南蛋白质折叠运输缺陷导致的内分泌及代谢疾病．目前已报道的由药物分子伴侣恢复功能的突变蛋白主要为质膜蛋白及细胞器蛋白,如ATP结合盒转运蛋白、G-蛋白耦联受体及溶酶体酶等．大量的细胞及动物实验结果显示了药物分子伴侣的临床应用前景广阔,目前已有一例临床实验获得了成功．     

Principles of protein folding, misfolding and aggregation

This review summarises our current understanding of the underlying and universal mechanism by which newly synthesised proteins achieve their biologically functional states. Protein molecules, however, all have a finite tendency either to misfold, or to fail to maintain their correctly folded states, under some circumstances. This article describes some of the consequences of such behaviour, particularly in the context of the aggregation events that are frequently associated with aberrant folding. It focuses in particular on the emerging links between protein aggregation and the increasingly prevalent forms of debilitating disease with which it is now known to be associated.     

The therapeutic potential of chemical chaperones in protein folding diseases

Several neurodegenerative diseases are caused by defects in protein folding, including Alzheimer, Parkinson, Huntington, and prion diseases. Once a disease-specific protein misfolds, it can then form toxic aggregates which accumulate in the brain, leading to neuronal dysfunction, cell death, and clinical symptoms. Although significant advances have been made toward understanding the mechanisms of protein aggregation, there are no curative treatments for any of these diseases. Since protein misfolding and the accumulation of aggregates are the most upstream events in the pathological cascade, rescuing or stabilizing the native conformations of proteins is an obvious therapeutic strategy. In recent years, small molecules known as chaperones have been shown to be effective in reducing levels of misfolded proteins, thus minimizing the accumulation of aggregates and their downstream pathological consequences. Chaperones are classified as molecular, pharmacological, or chemical. In this mini-review we summarize the modes of action of different chemical chaperones and discuss evidence for their efficacy in the treatment of protein folding diseases in vitro and in vivo.     

Pressure provides new insights into protein folding, dynamics and structure

Hydrostatic pressure is a powerful tool for studying protein folding, and the dynamics and structure of folding intermediates. Recently, pressure techniques have opened two important fronts to aid our understanding of how polypeptides fold into highly structured conformations. The first advance is the stabilization of folding intermediates, making it possible to characterize their structures and dynamics by different methodologies. Kinetic studies under pressure constitute the second advance, promising detailed appraisal and understanding of protein folding landscapes. The combination of these two approaches enables dissection of the roles of packing and cavities in folding, and in assembly of multimolecular structures such as protein-DNA complexes and viruses. The study of aggregates and amyloids, derived from partially folded intermediates at the junction between productive and off-pathway folding, have also been studied, promising better understanding of diseases associated with protein misfolding.     

Chaperone machines in action

How do chaperones operate in cells? For some major chaperones it is clear what they do, though mostly not how they do it. Hsp60, 70 and 100 families carry out folding, unfolding or disaggregation of proteins. Regarding mechanisms of action, we have the clearest picture of the ATP-driven mechanism of the bacterial Hsp60s, and structures of full-length Hsp70 and 90 family members are beginning to give insights into their allosteric mechanisms. Recent advances are giving an improved understanding of the nature of chaperone interactions with their non-native substrate proteins. There have also been significant advances in understanding the engagement of chaperones in preventing the formation of toxic aggregates in degenerative disease and the relationship of protein quality control to complex biological processes such as ageing.