Folding Dynamics of 10‐Residue β‐Hairpin Peptide Chignolin

Short peptides that fold into β‐hairpins are ideal model systems for investigating the mechanism of protein folding because their folding process shows dynamics typical of proteins. We performed folding, unfolding, and refolding molecular dynamics simulations (total of 2.7 μs) of the 10‐residue β‐hairpin peptide chignolin, which is the smallest β‐hairpin structure known to be stable in solution. Our results revealed the folding mechanism of chignolin, which comprises three steps. First, the folding begins with hydrophobic assembly. It brings the main chain together; subsequently, a nascent turn structure is formed. The second step is the conversion of the nascent turn into a tight turn structure along with interconversion of the hydrophobic packing and interstrand hydrogen bonds. Finally, the formation of the hydrogen‐bond network and the complete hydrophobic core as well as the arrangement of side‐chain–side‐chain interactions occur at approximately the same time. This three‐step mechanism appropriately interprets the folding process as involving a combination of previous inconsistent explanations of the folding mechanism of the β‐hairpin, that the first event of the folding is formation of hydrogen bonds and the second is that of the hydrophobic core, or vice versa.     

Refolding of urea‐denatured α‐chymotrypsin by protein‐folding liquid chromatography

An approach for re‐folding denatured proteins during proteome research by protein folding liquid chromatography (PFLC) is presented. Standard protein, α‐chymotrypsin (α‐Chy), was selected as a model protein and hydrophobic interaction chromatography was performed as a typical PFLC; the three different α‐Chy states – urea‐denatured (U state), its folded intermediates (M state) and nature state (N state) – were studied during protein folding. Based on the test by matrix‐assisted laser desorption/ionization time of flight mass spectrometry and bioactivity, only one stable M state of the α‐Chy was identified and then it was prepared for further investigation. The specific bioactivity of the refolded α‐Chy was found to be higher than that of commercial α‐Chy as the urea concentration in the sample solution ranged from 1.0 to 3.0 m ; the highest specific bioactivity at urea concentration was 1.0 m , indicating the possibility for re‐folding some proteins that have partially or completely lost their bioactivity, as a dilute urea solution was employed for dissolving the sample. The experiment showed that the peak height of its M state increased with increasing urea concentration, and correspondingly decreased in the amount of the refolded α‐Chy. When the urea concentration reached 6.0 m , the unfolded α‐Chy could not be refolded at all. Copyright © 2012 John Wiley & Sons, Ltd.     

Isomerization‐ and Temperature‐Jump‐Induced Dynamics of a Photoswitchable β‐Hairpin

Conformational changes in proteins and peptides can be initiated by diverse processes. This raises the question how the variation of initiation mechanisms is connected to differences in folding or unfolding processes. In this work structural dynamics of a photoswitchable β‐hairpin model peptide were initiated by two different mechanisms: temperature jump (T‐jump) and isomerization of a backbone element. In both experiments the structural changes were followed by time‐resolved IR spectroscopy in the nanosecond to microsecond range. When the photoisomerization of the azobenzene backbone switch initiated the folding reaction, pronounced absorption changes related to folding into the hairpin structure were found with a time constant of about 16 μs. In the T‐jump experiment kinetics with the same time constant were observed. For both initiation processes the reaction dynamics revealed the same strong dependence of the reaction time on temperature. The highly similar transients in the microsecond range show that the peptide dynamics induced by T‐jump and isomerization are both determined by the same mechanism and exclude a downhill‐folding process. Furthermore, the combination of the two techniques allows a detailed model for folding and unfolding to be presented: The isomerization‐induced folding process ends in a transition‐state reaction scheme, in which a high energetic barrier of 48 kJ mol^?1 separates unfolded and folded structures.     

Monitoring Backbone Hydrogen‐Bond Formation in β‐Barrel Membrane Protein Folding

β‐barrel membrane proteins are key components of the outer membrane of bacteria, mitochondria and chloroplasts. Their three‐dimensional structure is defined by a network of backbone hydrogen bonds between adjacent β‐strands. Here, we employ hydrogen–deuterium (H/D) exchange in combination with NMR spectroscopy and mass spectrometry to monitor backbone hydrogen bond formation during folding of the outer membrane protein X (OmpX) from E. coli in detergent micelles. Residue‐specific kinetics of interstrand hydrogen‐bond formation were found to be uniform in the entire β‐barrel and synchronized to formation of the tertiary structure. OmpX folding thus propagates via a long‐lived conformational ensemble state in which all backbone amide protons exchange with the solvent and engage in hydrogen bonds only transiently. Stable formation of the entire OmpX hydrogen bond network occurs downhill of the rate‐limiting transition state and thus appears cooperative on the overall folding time scale.     

Two‐Dimensional Crowding Uncovers a Hidden Conformation of α‐Synuclein

The intrinsically disordered protein (IDP), α‐synuclein (αS), is well‐known for phospholipid membrane binding‐coupled folding into tunable helical conformers. Here, using single‐molecule experiments in conjunction with ensemble assays and a theoretical model, we present a unique case demonstrating that the interaction–folding landscape of αS can be tuned by two‐dimensional (2D) crowding through simultaneous binding of a second protein on the bilayer surface. Unexpectedly, the experimental data show a clear deviation from a simple competitive inhibition model, but are consistent with a bimodal inhibition mechanism wherein membrane binding of a second protein (a membrane interacting chaperone, Hsp27, in this case) differentially inhibits two distinct modules of αS–membrane interaction. As a consequence, αS molecules are forced to access a hidden conformational state on the phospholipid bilayer in which only the higher‐affinity module remains membrane‐bound. Our results demonstrate that macromolecular crowding in two dimensions can play a significant role in shaping the conformational landscape of membrane‐binding IDPs with multiple binding modes.     

Dramatic Enhancement of Binding Affinities Between Foldamer‐Based Receptors and Anions by Intra‐Receptor π‐Stacking

As a synthetic model for intra‐protein interactions that reinforce binding affinities between proteins and ligands, the energetic interplay of binding and folding was investigated using foldamer‐based receptors capable of adopting helical structures. The receptors were designed to have identical hydrogen‐bonding sites for anion binding but different aryl appendages that simply provide additional π‐stacking within the helical backbones without direct interactions with the bound anions. In particular, the presence of electron‐deficient aryl appendages led to dramatic enhancements in the association constant between the receptor and chloride or nitrate ions, by up to three orders of magnitude. Extended stacking within the receptor contributes to the stabilization of the entire folding structure of complexes, thereby enhancing binding affinities.     

Direct Observation of the Reversible Two‐State Unfolding and Refolding of an α/β Protein by Single‐Molecule Atomic Force Microscopy

Directly observing protein folding in real time using atomic force microscopy (AFM) is challenging. Here the use of AFM to directly monitor the folding of an α/β protein, NuG2, by using low‐drift AFM cantilevers is demonstrated. At slow pulling speeds (<50 nm s^?1), the refolding of NuG2 can be clearly observed. Lowering the pulling speed reduces the difference between the unfolding and refolding forces, bringing the non‐equilibrium unfolding–refolding reactions towards equilibrium. At very low pulling speeds (ca. 2 nm s^?1), unfolding and refolding were observed to occur in near equilibrium. Based on the Crooks fluctuation theorem, we then measured the equilibrium free energy change between folded and unfolded states of NuG2. The improved long‐term stability of AFM achieved using gold‐free cantilevers allows folding–unfolding reactions of α/β proteins to be directly monitored near equilibrium, opening the avenue towards probing the folding reactions of other mechanically important α/β and all‐β elastomeric proteins.     

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.     

Single molecule force spectroscopy using polyproteins

In recent years single molecule force spectroscopy has emerged as a powerful new tool to explore the mechanical stability and folding pathways of individual proteins. This technique is used to apply a stretching force between two points of a protein, unfolding the protein to an extended state. By measuring the unfolding and folding trajectories of individual proteins, insight can be gained into the physical mechanisms of protein folding. In this tutorial review we introduce the reader to single molecule force spectroscopy using the atomic force microscope (AFM), and explain the two main modes of operation of the AFM for force spectroscopy: force-extension and force-clamp. We introduce the approach of using polyproteins to obtain a clear mechanical fingerprint for monitoring the response of proteins to an applied mechanical force. In addition, we provide an informative and representative review of recent research on proteins using single molecule force spectroscopy. We focus on areas which have made a significant contribution to the single molecule protein folding field and highlight emerging areas of research which have wider implications for the general scientific community.     

Oxime Side‐Chain Cross‐Links in an α‐Helical Coiled‐Coil Protein: Structure,Thermodynamics, and Folding‐Templated Synthesis of Bicyclic Species

Covalent side‐chain cross‐links are a versatile method to control peptide folding, particularly when α‐helical secondary structure is the target. Here, we examine the application of oxime bridges, formed by the chemoselective reaction between aminooxy and aldehyde side chains, for the stabilization of a helical peptide involved in a protein–protein complex. A series of sequence variants of the dimeric coiled coil GCN4‐p1 bearing oxime bridges at solvent‐exposed positions were prepared and biophysically characterized. Triggered unmasking of a side‐chain aldehyde in situ and subsequent cyclization proceed rapidly and cleanly at pH 7 in the folded protein complex. Comparison of folding thermodynamics among a series of different oxime bridges show that the cross links are consistently stabilizing to the coiled coil, with the extent of stabilization sensitive to the exact size and structure of the macrocycle. X‐ray crystallographic analysis of a coiled coil with the best cross link in place and a second structure of its linear precursor show how the bridge is accommodated into an α‐helix. Preparation of a bicyclic oligomer by simultaneous formation of two linkages in situ demonstrates the potential use of triggered oxime formation to both trap and stabilize a particular peptide folded conformation in the bound state.     

Filaggrin Peptides with β‐Hairpin Structure Bind Rheumatoid Arthritis Antibodies

In the early detection of rheumatoid arthritis (RA) synthetic filaggrin peptides serve as antigens for rheumatoid‐specific autoantibodies (anti‐citrullinated peptide antibody, ACPA) in ELISA tests. In this work we present a peptide that exhibits the binding epitope of ACPA in the form of a stable folding β‐hairpin. The homogeneity of the peptide folding was confirmed by NMR spectroscopy and might lead to the first proposed structure of the antibody‐bound conformation of the epitope.     

Chaperonin GroEL: structure and reaction cycle

The structure of Escherichia coli chaperonin GroEL was studied using various experimental tools. Such studies produced information about its structure with increasing details. Moreover, remarkable advances in experimental methods provided a step forward in understanding the reaction cycle involved in GroEL-mediated protein folding. In the current review we summarize recent progress, focus on the structure of GroEL and understand the mechanism involved in GroEL-mediated protein folding. This review is divided into the following sections: (i) Section 1 provides basic understanding on protein folding, (ii) Section 2 not only describes various tools used to elucidate the structural aspects of GroEL but also provides details about its structure with particular emphasis, (iii) Section 3 describes allosteric transitions and the reaction cycle involved in GroEL-mediated protein folding, (iv) Section 4 explains iterative annealing and smoothing of the energy landscape model and finally (v) Section 5 discusses applications and recent progress.     

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.     

Cytochrome‐P450‐Induced Ordering of Microsomal Membranes Modulates Affinity for Drugs

Although membrane environment is known to boost drug metabolism by mammalian cytochrome P450s, the factors that stabilize the structural folding and enhance protein function are unclear. In this study, we use peptide‐based lipid nanodiscs to “trap” the lipid boundaries of microsomal cytochrome P450 2B4. We report the first evidence that CYP2B4 is able to induce the formation of raft domains in a biomimetic compound of the endoplasmic reticulum. NMR experiments were used to identify and quantitatively determine the lipids present in nanodiscs. A combination of biophysical experiments and molecular dynamics simulations revealed a sphingomyelin binding region in CYP2B4. The protein‐induced lipid raft formation increased the thermal stability of P450 and dramatically altered ligand binding kinetics of the hydrophilic ligand BHT. These results unveil membrane/protein dynamics that contribute to the delicate mechanism of redox catalysis in lipid membrane.     

The 3′-End of tRNA and Its Role in Protein Biosynthesis

In the course of protein biosynthesis, the 3′-ends of aminoacyl-tRNA (aa-tRNA) and peptidyl-tRNA specifically interact with macromolecules of the protein biosynthesis machinery. The 3′-end of tRNA consists of an invariant C-C-A single strand. Interaction of the aminoacyl-tRNA 3′-end with elongation factor Tu (EF-Tu) containing bound GTP is necessary for the formation of the aa-tRNA·EF-Tu·GTP complex and, after the complex binds to the ribosome, for the GTP hydrolysis. This process is followed by the specific binding of the aminoacyl-tRNA 3′-end to the aminoacyl (A) site of the ribosome. In this review, a model is proposed that involves Watson-Crick base pairing of the C? C sequence of the aminoacyl-tRNA 3′-end with a specific G? G sequence of the ribosomal 23S RNA. Similarly, peptidyl-tRNA binds with its 3′-end to the peptidyl (P) site of the ribosome. This binding may also involve Watson-Crick base pairing of the C-C-A sequence with a complementary sequence of 23S RNA. It is proposed that peptide bond formation is catalyzed by a functional site of the 23S RNA located near the 3′-ends of aminoacyl-tRNA and peptidyl-tRNA. A model is suggested in which two loops of the 23S RNA, brought into close proximity via folding, are involved both in binding the 3′-ends of the tRNAs and in catalyzing peptide bond formation. This model presumes a dynamic structure for ribosomal RNA, which is modulated by interaction with elongation factors and ribosomal proteins.     

Forty years of research on osmolyte-induced protein folding and stability

Most organisms that have adapted to environmental stresses have done so by production and accumulation of certain small organic molecules, known as osmolytes that arose by natural selection and have the ability to stabilize intracellular proteins against the environmental stress. It is well known that osmolytes stabilize proteins and induce folding of aberrant proteins and therefore, it is of therapeutic use for a large number of protein misfolding diseases. Thus, it is very important that the present knowledge of the ability and mechanism of osmolyte-induced protein folding and structural stabilization should reach to researchers working in different avenues. In around 40 years of research, we have gained great advances in various aspects of protein folding and structural stabilization induced by osmolytes. To summarize and discuss the original findings, many short review articles and few long reviews have also been available but almost all have focuses on specific aspects. To get a clear picture of the effect of osmolytes on protein folding and structural stabilization, it is necessary for the benefits of the general readers, to combine and discuss all findings made during its 40 years of life. This review article is therefore, designed to give a collective knowledge on almost all facets of the progresses made on osmolyte-protein interaction to-date.     

Monitoring copopulated conformational states during protein folding events using electrospray ionization-ion mobility spectrometry-mass spectrometry

The precise mechanism of protein folding remains elusive and there is a deficiency of biophysical techniques that are capable of monitoring the individual behavior of copopulated protein conformers during the folding process. Herein, an ion mobility spectrometry (IMS) device integrated with electrospray ionization mass spectrometry (ESI-MS) has been used to successfully separate and analyze protein conformers differing in cross section and/or charge state. In an initial test, an ensemble of folded and partially folded conformers of the protein cytochrome c was separated. A detailed study undertaken on the amyloidogenic protein beta(2)-microglobulin (beta(2)m), which forms fibrils by protein unfolding followed by self-aggregation and is responsible for the disease dialysis-related amyloidosis, has generated important insights into its folding landscape. Initially, a systematic titration of beta(2)m over the pH range 2 to 7 using ESI-IMS-MS allowed individual conformers to be monitored and quantified throughout the acid denaturation process. Furthermore, a comparison of wild-type beta(2)m with single and double amino acid variants with a range of folding stabilities and propensities for amyloid fibril formation has provided illuminating evidence of the role of different conformers in protein stability and amyloidogenic aggregation. The ESI-IMS-MS data presented here not only demonstrate an important and informative further dimension to ESI-MS, but also illustrate the potential of the ESI-IMS-MS technique for unravelling protein folding enigmas in general and studying protein misfolding diseases in particular.     

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.     

The key role of membranes in amyloid formation from a biophysical perspective

Even though our knowledge of how proteins misfold and aggregate is deeper nowadays, the mechanisms driving this process are still poorly understood. Among the factors involved, membranes should be taken into account. Indeed, convincing evidence suggests that membranes may influence protein folding, misfolding and aggregation. In fact, membrane lipid composition of different cellular types may attenuate or intensify the environmental pressure over protein folding equilibrium. In the present review the aim is to make an up-to-date analysis of the membrane influence on protein aggregation from a biophysical point of view in order to provide useful tools for researchers from other fields. In particular, we discuss how membranes can alter protein environment, e.g. increasing local protein concentration, lowering pH and dielectric constant, allowing accessibility to the hydrophobic milieu and promoting surface crowding, all of which will lead to protein aggregation. In addition, we review the role that specific lipids may exert on protein aggregation and finally we analyse the possible implication of membrane-related oxidative stress on amyloidogenesis.     

The Bright Future of Unconventional σ/π‐Hole Interactions

Non‐covalent interactions play a crucial role in (supramolecular) chemistry and much of biology. Supramolecular forces can indeed determine the structure and function of a host–guest system. Many sensors, for example, rely on reversible bonding with the analyte. Natural machineries also often have a significant non‐covalent component (e.g. protein folding, recognition) and rational interference in such ‘living’ devices can have pharmacological implications. For the rational design/tweaking of supramolecular systems it is helpful to know what supramolecular synthons are available and to understand the forces that make these synthons stick to one another. In this review we focus on σ‐hole and π‐hole interactions. A σ‐ or π‐hole can be seen as positive electrostatic potential on unpopulated σ* or π⁽*⁾ orbitals, which are thus capable of interacting with some electron dense region. A σ‐hole is typically located along the vector of a covalent bond such as X?H or X?Hlg (X=any atom, Hlg=halogen), which are respectively known as hydrogen and halogen bond donors. Only recently it has become clear that σ‐holes can also be found along a covalent bond with chalcogen (X?Ch), pnictogen (X?Pn) and tetrel (X?Tr) atoms. Interactions with these synthons are named chalcogen, pnigtogen and tetrel interactions. A π‐hole is typically located perpendicular to the molecular framework of diatomic π‐systems such as carbonyls, or conjugated π‐systems such as hexafluorobenzene. Anion–π and lone‐pair–π interactions are examples of named π‐hole interactions between conjugated π‐systems and anions or lone‐pair electrons respectively. While the above nomenclature indicates the distinct chemical identity of the supramolecular synthon acting as Lewis acid, it is worth stressing that the underlying physics is very similar. This implies that interactions that are now not so well‐established might turn out to be equally useful as conventional hydrogen and halogen bonds. In summary, we describe the physical nature of σ‐ and π‐hole interactions, present a selection of inquiries that utilise σ‐ and π‐holes, and give an overview of analyses of structural databases (CSD/PDB) that demonstrate how prevalent these interactions already are in solid‐state structures.