蛋白质体外折叠技术研究现状与展望

蛋白质折叠技术是生物工程的新“单元操作”。本文阐述了蛋白质体外折叠研究的重要理论意义和应用价值，综述了目前蛋白质体外折叠技术研究进展，最后对蛋白质折叠技术的研究进行了展望。     

蛋白质折叠与淀粉样沉积的产生

蛋白质是生物体内一切功能的执行者,而蛋白质正确折叠则是发挥其生理功能的一个重要前提条件。近年来研究发现,蛋白质的错误折叠可以形成淀粉样沉积进而导致一些疾病的产生。探讨了蛋白质折叠和淀粉样纤维产生的机制。     

Protein Folding and Misfolding on Surfaces

Protein folding, misfolding and aggregation, as well as the way misfolded and aggregated proteins affects cell viability are emerging as key themes in molecular and structural biology and in molecular medicine. Recent advances in the knowledge of the biophysical basis of protein folding have led to propose the energy landscape theory which provides a consistent framework to better understand how a protein folds rapidly and efficiently to the compact, biologically active structure. The increased knowledge on protein folding has highlighted its strict relation to protein misfolding and aggregation, either process being in close competition with the other, both relying on the same physicochemical basis. The theory has also provided information to better understand the structural and environmental factors affecting protein folding resulting in protein misfolding and aggregation into ordered or disordered polymeric assemblies. Among these, particular importance is given to the effects of surfaces. The latter, in some cases make possible rapid and efficient protein folding but most often recruit proteins/peptides increasing their local concentration thus favouring misfolding and accelerating the rate of nucleation. It is also emerging that surfaces can modify the path of protein misfolding and aggregation generating oligomers and polymers structurally different from those arising in the bulk solution and endowed with different physical properties and cytotoxicities.     

采用二态模型研究邻苯二甲酸酐修饰对HRP热变性的影响

本研究采用二态假说模型,利用热力学原理从宏观角度对辣根过氧化物酶（HRP）的热变性进行研究,通过相关的计算得到表征该酶蛋白变性的热力学参数：变性自由能、变性焓、变性熵和解链温度．研究结果显示,邻苯二甲酸酐修饰后的HRP蛋白酶的解链温度为692℃,高于天然HRP的懈链温度622℃,反映了邻苯二酸酐修饰可以明显的提高HRP的热稳定性。     

Protein Folding and Mechanisms of Proteostasis

Highly sophisticated mechanisms that modulate protein structure and function, which involve synthesis and degradation, have evolved to maintain cellular homeostasis. Perturbations in these mechanisms can lead to protein dysfunction as well as deleterious cell processes. Therefore in recent years the etiology of a great number of diseases has been attributed to failures in mechanisms that modulate protein structure. Interconnections among metabolic and cell signaling pathways are critical for homeostasis to converge on mechanisms associated with protein folding as well as for the preservation of the native structure of proteins. For instance, imbalances in secretory protein synthesis pathways lead to a condition known as endoplasmic reticulum (ER) stress which elicits the adaptive unfolded protein response (UPR). Therefore, taking this into consideration, a key part of this paper is developed around the protein folding phenomenon, and cellular mechanisms which support this pivotal condition. We provide an overview of chaperone protein function, UPR via, spatial compartmentalization of protein folding, proteasome role, autophagy, as well as the intertwining between these processes. Several diseases are known to have a molecular etiology in the malfunction of mechanisms responsible for protein folding and in the shielding of native structure, phenomena which ultimately lead to misfolded protein accumulation. This review centers on our current knowledge about pathways that modulate protein folding, and cell responses involved in protein homeostasis.     

Rise-Time of FRET-Acceptor Fluorescence Tracks Protein Folding

Uniform labeling of proteins with fluorescent donor and acceptor dyes with an equimolar ratio is paramount for accurate determination of Förster resonance energy transfer (FRET) efficiencies. In practice, however, the labeled protein population contains donor-labeled molecules that have no corresponding acceptor. These FRET-inactive donors contaminate the donor fluorescence signal, which leads to underestimation of FRET efficiencies in conventional fluorescence intensity and lifetime-based FRET experiments. Such contamination is avoided if FRET efficiencies are extracted from the rise time of acceptor fluorescence upon donor excitation. The reciprocal value of the rise time of acceptor fluorescence is equal to the decay rate of the FRET-active donor fluorescence. Here, we have determined rise times of sensitized acceptor fluorescence to study the folding of double-labeled apoflavodoxin molecules and show that this approach tracks the characteristics of apoflavodoxinʼs complex folding pathway.     

Going Non-native To Improve Oxidative Protein Folding

Chemical origami: Protein folding rates and yields can be limited by the presence of kinetic traps. By replacing two cysteines with selenocysteines in bovine pancreatic trypsin inhibitor, Hilvert and co-workers increased the folding rate and removed two traps from the oxidative-folding pathway, even though the resulting diselenide would correspond to a non-native disulfide bond.     

Protein GB1 Folding and Assembly from Structural Elements

Folding of the Protein G B1 domain (PGB1) shifts with increasing salt concentration from a cooperative assembly of inherently unstructured subdomains to an assembly of partly pre-folded structures. The salt-dependence of pre-folding contributes to the stability minimum observed at physiological salt conditions. Our conclusions are based on a study in which the reconstitution of PGB1 from two fragments was studied as a function of salt concentrations and temperature using circular dichroism spectroscopy. Salt was found to induce an increase in β-hairpin structure for the C-terminal fragment (residues 41 – 56), whereas no major salt effect on structure was observed for the isolated N-terminal fragment (residues 1 – 41). In line with the increasing evidence on the interrelation between fragment complementation and stability of the corresponding intact protein, we also find that salt effects on reconstitution can be predicted from salt dependence of the stability of the intact protein. Our data show that our variant (which has the mutations T2Q, N8D, N37D and reconstitutes in a manner similar to the wild type) displays the lowest equilibrium association constant around physiological salt concentration, with higher affinity observed both at lower and higher salt concentration. This corroborates the salt effects on the stability towards denaturation of the intact protein, for which the stability at physiological salt is lower compared to both lower and higher salt concentrations. Hence we conclude that reconstitution reports on molecular factors that govern the native states of proteins.     

The Folding of de Novo Designed Protein DS119 via Molecular Dynamics Simulations

As they are not subjected to natural selection process, de novo designed proteins usually fold in a manner different from natural proteins. Recently, a de novo designed mini-protein DS119, with a βαβ motif and 36 amino acids, has folded unusually slowly in experiments, and transient dimers have been detected in the folding process. Here, by means of all-atom replica exchange molecular dynamics (REMD) simulations, several comparably stable intermediate states were observed on the folding free-energy landscape of DS119. Conventional molecular dynamics (CMD) simulations showed that when two unfolded DS119 proteins bound together, most binding sites of dimeric aggregates were located at the N-terminal segment, especially residues 5–10, which were supposed to form β-sheet with its own C-terminal segment. Furthermore, a large percentage of individual proteins in the dimeric aggregates adopted conformations similar to those in the intermediate states observed in REMD simulations. These results indicate that, during the folding process, DS119 can easily become trapped in intermediate states. Then, with diffusion, a transient dimer would be formed and stabilized with the binding interface located at N-terminals. This means that it could not quickly fold to the native structure. The complicated folding manner of DS119 implies the important influence of natural selection on protein-folding kinetics, and more improvement should be achieved in rational protein design.     

Expression Characterization of AtPDI11 and Functional Analysis of AtPDI11 D Domain in Oxidative Protein Folding

The formation and isomerization of disulfide bonds mediated by protein disulfide isomerase (PDI) in the endoplasmic reticulum (ER) is of fundamental importance in eukaryotes. Canonical PDI structure comprises four domains with the order of a-b-b′-a′. In Arabidopsis thaliana, the PDI-S subgroup contains only one member, AtPDI11, with an a-a′-D organization, which has no orthologs in mammals or yeast. However, the expression pattern of AtPDI11 and the functioning mechanism of AtPDI11 D domain are currently unclear. In this work, we found that PDI-S is evolutionarily conserved between land plants and algal organisms. AtPDI11 is expressed in various tissues and its induction by ER stress is disrupted in bzip28/60 and ire1a/b mutants that are null mutants of key components in the unfolded protein response (UPR) signal transduction pathway, suggesting that the induction of AtPDI11 by ER stress is mediated by the UPR signaling pathway. Furthermore, enzymatic activity assays and genetic evidence showed that the D domain is crucially important for the activities of AtPDI11. Overall, this work will help to further understand the working mechanism of AtPDI11 in catalyzing disulfide formation in plants.     

蛋白质折叠及二级结构预测

蛋白质二级结构预测是研究蛋白质折叠的主要内容之一,也是获得新氨基酸序列结构信息的一般方法。从热力学和动力学两方面对蛋白质折叠机理进行分析,对蛋白质二级结构预测的常用方法进行分析和评价,并提出蛋白质空间结构预测的途径。     

Protein Design with Fluoroprolines: 4,4-Difluoroproline Does Not Eliminate the Rate-Limiting Step of Thioredoxin Folding

C⁴-substituted fluoroprolines (4R)-fluoroproline ((4R)-Flp) and (4S)-fluoroproline ((4S)-Flp) have been used in protein engineering to enhance the thermodynamic stability of peptides and proteins. The electron-withdrawing effect of fluorine can bias the pucker of the pyrrolidine ring, influence the conformational preference of the preceding peptide bond, and can accelerate the cis/trans prolyl peptide bond isomerisation by diminishing its double bond character. The role of 4,4-difluoroproline (Dfp) in the acceleration of the refolding rate of globular proteins bearing a proline (Pro) residue in the cis conformation in the native state remains elusive. Moreover, the impact of Dfp on the thermodynamic stability and bioactivity of globular proteins has been seldom described. In this study, we show that the incorporation of Dfp caused a redox state dependent and position dependent destabilisation of the thioredoxin (Trx) fold, while the catalytic activities of the modified proteins remained unchanged. The Pro to Dfp substitution at the conserved cisPro76 in the thioredoxin variant Trx1P did not elicited acceleration of the rate-limiting trans-to-cis isomerization of the Ile75-Pro76 peptide bond. Our results show that pucker preferences in the context of a tertiary structure could play a major role in protein folding, thus overtaking the rules determined for cis/trans isomerisation barriers determined in model peptides.     

Selenoglutathione-Mediated Rescue of Kinetically Trapped Intermediates in Oxidative Protein Folding

Selenoglutathione has been shown to have considerable potential as a catalyst of oxidative protein folding. Here we examine how this reagent modulates the folding pathway of bovine pancreatic trypsin inhibitor (BPTI) and show that the diselenide increases the efficiency of this process primarily by accelerating the conversion of a kinetically trapped folding intermediate.     

Folding of Trp-cage Mini Protein Using Temperature and Biasing Potential Replica��Exchange Molecular Dynamics Simulations

The folding process of the 20 residue Trp-cage mini-protein was investigated using standard temperature replica exchange molecular dynamics (T-RexMD) simulation and a biasing potential RexMD (BP-RexMD) method. In contrast to several conventional molecular dynamics simulations, both RexMD methods sampled conformations close to the native structure after 10–20 ns simulation time as the dominant conformational states. In contrast, to T-RexMD involving 16 replicas the BP-RexMD method achieved very similar sampling results with only five replicas. The result indicates that the BP-RexMD method is well suited to study folding processes of proteins at a significantly smaller computational cost, compared to T-RexMD. Both RexMD methods sampled not only similar final states but also agreed on the sampling of intermediate conformations during Trp-cage folding. The analysis of the sampled potential energy contributions indicated that Trp-cage folding is favored by both van der Waals and to a lesser degree electrostatic contributions. Folding does not introduce any significant sterical strain as reflected by similar energy distributions of bonded energy terms (bond length, bond angle and dihedral angle) of folded and unfolded Trp-cage structures.     

Folding of Right- and Left-Handed Three-Helix Proteins

We are the first to investigate the relationship between protein handedness and the rate of protein folding. Our findings demonstrate that small three-helix, left-handed proteins are less densely packed and should result in faster folding than that of right-handed, three-helix proteins. At the same time, right-handed, three-helix proteins have higher mechanical stability than the left-handed proteins. Moreover, from our analysis we have revealed that bacterial three-helix proteins have some advantages in packing over eukaryotic right-handed, three-helix proteins, which should result in faster folding.     

From Side Chains Rattling on Picoseconds to Ensemble Simulations of Protein Folding

Simulations of biological macromolecules have evolved tremendously since the discoveries of the 1970s. The field has moved from simple simulations in vacuo on picosecond scales to milliseconds of accurate sampling of large proteins, and it has become a standard tool in biochemistry and biophysics, rather than a dedicated theoretical one. This is partly due to increasing computational power, but it would not have been possible without huge research efforts invested in new algorithms and software. Here, we illustrate some of this development, both past and future challenges, and in particular, discuss how the recent introduction of modern ensemble methods is breaking the trend of ever-longer simulations to instead focus on throughput and sampling. This has not only helped simulations become much more accurate, but it provides statistical error estimates, which are critical, as simulations are increasingly used to predict properties that have not yet been measured experimentally.     

Relationship between Changes in the Protein Folding Pathway and the Process of Amyloid Formation: The Case of Bovine Carbonic Anhydrase II

Many proteins form amyloid fibrils only under conditions when the probability of transition from a native (structured, densely packed) to an intermediate (labile, destabilized) state is increased. It implies the assumption that some structural intermediates are more convenient for amyloid formation than the others. Hence, if a mutation affects the protein folding pathway, one should expect that this mutation could affect the rate of amyloid formation as well. In the current work, we have compared the effects of amino acid substitutions of bovine carbonic anhydrase II on its unfolding pathway and on its ability to form amyloids at acidic pH and an elevated temperature. Wild-type protein and four mutant forms (L78A, L139A, I208A, and M239A) were studied. We analyzed the change of the protein unfolding pathway by the time-resolved fluorescence technique and the process of amyloid formation by thioflavin T fluorescence assay and electron microscopy. It was revealed that I208A substitution accelerates amyloid formation and affects the structure of the late (molten globule-like)-intermediate state of carbonic anhydrase, whereas the other mutations slow down the growth of amyloids and have either no effect on the unfolding pathway (L78A, L139A) or alter the conformational states arising at the early unfolding stage (M239A).     

Protein Folding in the Presence of Water‐Soluble Cyclic Diselenides with Novel Oxidoreductase and Isomerase Activities

The protein disulfide isomerase (PDI) family, found in the endoplasmic reticulum (ER) of the eukaryotic cell, catalyzes the formation and cleavage of disulfide bonds and thereby helps in protein folding. A decrease in PDI activity under ER stress conditions leads to protein misfolding, which is responsible for the progression of various human diseases, such as Alzheimer's, Parkinson's, diabetes mellitus, and atherosclerosis. Here we report that water‐soluble cyclic diselenides mimic the multifunctional activity of the PDI family by facilitating oxidative folding, disulfide formation/reduction, and repair of the scrambled disulfide bonds in misfolded proteins.     

Arrow of Time,Entropy, and Protein Folding: Holistic View on Biochirality

Chirality is a universal phenomenon, embracing the space–time domains of non-organic and organic nature. The biological time arrow, evident in the aging of proteins and organisms, should be linked to the prevalent biomolecular chirality. This hypothesis drives our exploration of protein aging, in relation to the biological aging of an organism. Recent advances in the chirality discrimination methods and theoretical considerations of the non-equilibrium thermodynamics clarify the fundamental issues, concerning the biphasic, alternative, and stepwise changes in the conformational entropy associated with protein folding. Living cells represent open, non-equilibrium, self-organizing, and dissipative systems. The non-equilibrium thermodynamics of cell biology are determined by utilizing the energy stored, transferred, and released, via adenosine triphosphate (ATP). At the protein level, the synthesis of a homochiral polypeptide chain of L-amino acids (L-AAs) represents the first state in the evolution of the dynamic non-equilibrium state of the system. At the next step the non-equilibrium state of a protein-centric system is supported and amended by a broad set of posttranslational modifications (PTMs). The enzymatic phosphorylation, being the most abundant and ATP-driven form of PTMs, illustrates the principal significance of the energy-coupling, in maintaining and reshaping the system. However, the physiological functions of phosphorylation are under the permanent risk of being compromised by spontaneous racemization. Therefore, the major distinct steps in protein-centric aging include the biosynthesis of a polypeptide chain, protein folding assisted by the system of PTMs, and age-dependent spontaneous protein racemization and degradation. To the best of our knowledge, we are the first to pay attention to the biphasic, alternative, and stepwise changes in the conformational entropy of protein folding. The broader view on protein folding, including the impact of spontaneous racemization, will help in the goal-oriented experimental design in the field of chiral proteomics.