胰岛素的体外折叠: 折叠中间体的鉴定 及其折叠途径

在单链胰岛素体外折叠研究的基础上, 研究了胰岛素的体外折叠过程. 在胰岛素的折叠过程中捕捉到6个主要的折叠中间体, 分别命名为P1A, P2B, P3A, P4B, P5B和P6B. 中间体的折叠实验证明6个中间体都能最终折叠成胰岛素. 在中间体的鉴定和分析以及推断的过渡态中间体形成和作用的基础上, 提出了胰岛素体外折叠的两步折叠途径. (ⅰ) 在折叠过程的早期, 完全还原的胰岛素, 即A链和B链各自形成中间体: 2个A链中间体(P1A和P3A), 4个B链中间体(P2B, P4B, P5B和P6B). (ⅱ) 在进一步的折叠过程中, 推断相继产生3个过渡态中间体: 首先, 中间体P3A通过分子内巯基/二硫键交换反应形成过渡态中间体Ⅰ, 该中间体含有1个天然二硫键A6-A11和2个巯基分别位于A7和A20; 然后, 过渡态中间体Ⅰ的巯基和P4B或P6B上的巯基通过氧化反应(主要以折叠体系中的GSSG为氧化剂)分别形成含有2个天然二硫键的过渡态中间体Ⅱ和Ⅲ; 最后, 过渡态中间体Ⅱ和Ⅲ通过分子内巯基/二硫键交换反应形成第3个天然二硫键, 完成胰岛素体外折叠.     

胰岛素的体外折叠: 折叠中间体的鉴定 及其折叠途径

在单链胰岛素体外折叠研究的基础上, 研究了胰岛素的体外折叠过程. 在胰岛素的折叠过程中捕捉到6个主要的折叠中间体, 分别命名为P1A, P2B, P3A, P4B, P5B和P6B. 中间体的折叠实验证明6个中间体都能最终折叠成胰岛素. 在中间体的鉴定和分析以及推断的过渡态中间体形成和作用的基础上, 提出了胰岛素体外折叠的两步折叠途径. (ⅰ) 在折叠过程的早期, 完全还原的胰岛素, 即A链和B链各自形成中间体: 2个A链中间体(P1A和P3A), 4个B链中间体(P2B, P4B, P5B和P6B). (ⅱ) 在进一步的折叠过程中, 推断相继产生3个过渡态中间体: 首先, 中间体P3A通过分子内巯基/二硫键交换反应形成过渡态中间体Ⅰ, 该中间体含有1个天然二硫键A6-A11和2个巯基分别位于A7和A20; 然后, 过渡态中间体Ⅰ的巯基和P4B或P6B上的巯基通过氧化反应(主要以折叠体系中的GSSG为氧化剂)分别形成含有2个天然二硫键的过渡态中间体Ⅱ和Ⅲ; 最后, 过渡态中间体Ⅱ和Ⅲ通过分子内巯基/二硫键交换反应形成第3个天然二硫键, 完成胰岛素体外折叠.     

重组单链胰岛素在含有巯基试剂的变性剂中的解折叠

重组单链胰岛素(PIP)含有3对二硫键。在含有巯基试剂的变性剂中,PIP产生二硫键交换从而形成一系列具有不同解折叠程度的二硫键异构体混合物。分别用高压液相色谱（HPLC）和圆二色性（CD）光谱分析了PIP在含有0．2mmol/L2－巯基乙醇的尿素和盐酸胍中的解中的解折叠程度。PIP二硫键异构体混合物通过胰蛋白酶酶解并用质谱测定酶解片段的分子量,证明PIP确实产生了二硫键交换。同时还分离纯化了PIP的一种主要非天然二硫键异构体并研究了它重新折叠成天然构象的情况。观察到PIP只有一种热力学稳定的二硫键配对方式,PIP的非天然二硫键异构体在巯基试剂存在的条件下可以高效转化为天然二硫键配对。还将PIP解折叠和再折叠的情况与胰岛素样生长因子－I（IGF－I）及胰岛素做了比较：胰岛素和PIP只折叠成一种热力学稳定的三级结构,IGF－I却折叠成两种热力学稳定的二硫键异构体;胰岛素的双链重组需缓慢进行,而PIP却可以快速折叠。     

类胰岛素生长因子-1的C结构域不影响胰岛素二硫键的热力学稳定性

胰岛素和类胰岛素生长因子-1(IGF-1)都属于胰岛素超家族,两者不但一级、三级结构有较高的同源性,而且生理功能也有少量交叉.然而两者的折叠行为却有很大的差别：胰岛素及其重组前体(PIP)只折叠成一种热力学稳定的二硫键配对,而IGF-1却折叠成两种热力学稳定的二硫键异构体.为了了解两者折叠行为差异的分子机制,制备了由胰岛素的A,B链和IGF-1的C结构域构成的单链杂交分子——[B10Glu]Ins/IGF-1(C),研究了该杂交分子二硫键的热力学稳定性以及它在含有少量巯基试剂的变性剂中的解折叠程度,同时还纯化了该杂交分子一种主要的非天然二硫键异构体,并研究了它的再折叠情况. 观察到IGF-1中C结构域的引入并未改变胰岛素分子的折叠热力学,但是影响了折叠的动力学过程.     

基于氢氘交换核磁共振技术研究蛋白质与阳离子交换介质的相互作用

原位实时捕捉多孔介质内的蛋白结构变化信息是蛋白质层析失活机理研究中的难点。为此,本文发展了蛋白质氢氘交换与核磁共振(Nuclear magnetic resonance,NMR)相结合的新型蛋白质液固界面表征路线。研究了溶菌酶在溶液态以及在阳离子交换介质内部吸附态时的去折叠行为,并揭示了蛋白与阳离子交换介质(SP Sepharose FF)相互作用机理。溶液态溶菌酶的一维核磁共振氢谱动力学显示蛋白质去折叠可导致残基暴露进而加快氢氘交换速率。吸附态溶菌酶的二维氢-氢全相关谱图(Total correlation spectroscopy,TOCSY)以及残基峰强度显示,溶菌酶在吸附态时的去折叠呈区域性,无规则卷曲(Coil,bend,and turn)片段的酰胺氢信号更容易失去,而二级结构域(α-helix,β-sheet)对酰胺氢信号保护更好。最终,利用蛋白表面静电势模拟计算结合氢氘标记的蛋白核核磁数据可确定出溶菌酶与阳离子交换介质的作用位点。这对于深刻理解层析过程中蛋白与层析介质微观作用机理以及层析过程中吸附剂的选择、设计具有重要意义,也为获取蛋白质与生物材料之间相互作用研究提供新的有效工具。     

分子伴侣(MolecularChaperones)与蛋白质的折叠

尽管几百种蛋白质的三维结构已研究得非常清楚,但这些蛋白质折叠成其天然构象的机制与途径仍有待于进一步的研究。有关于蛋白质折叠的一些体外实验确定,决定一个蛋白质三维结构的信息存在于蛋白质的多肽链之中。在体外,一定条件下,在没有任何其他蛋白质存更多还原在下,一些酶和蛋白质可以自我装配成天然构象[1,2].     

组蛋白和核酸的1~H、(31)~P核磁共振研究

本文报道了组蛋白、DNA、聚核苷酸A和聚核苷酸U的~1H、~（31）P高分辨核磁共振研究新结果。 当组蛋白与DNA、聚核苷酸A或U相互作用后,核酸的质子峰变弱或消失。     

不同年龄红细胞(人)的增溶胰岛素受体和胰岛素结合以及依赖于胰岛素的磷酸化反应

1.我们研究了人的不同年龄红细胞的胰岛素受体从膜上增溶后和胰岛素相结合的特征。结果表明,随着红细胞年龄的增加,增溶胰岛素受体和胰岛素的最大结合能力下降;ED_(50)降低,同时受体结合位点数减少。青年红细胞的增溶胰岛素受体数比去年的高一倍,但其亲和性不论在结合平衡状态或动力学状态都基本一致。 2.利用γ-~(32)P-ATP的~(32)P参入量研究了胰岛素对不同年龄红细胞增溶蛋白质及外源蛋白质的磷酸化的影响。在胰岛素存在时,磷酸化分别增加2.3—2.9倍,及3.4—4.4倍。胰岛素刺激青年红细胞的增溶蛋白质的磷酸化的敏感性为老年的2.4倍。 3.研究结果表明,单个人的血样可用来研究胰岛素受体的结合及激酶的性质。     

A7—B7二硫键缺失对胰岛素原重折叠及结构的影响

为研究A7 B7二硫键在胰岛素原结构和折叠中的作用 ,构建了A7 B7二硫键缺失的胰岛素原突变体 ,研究了其与野生型胰岛素原在体外重折叠产率、自由巯基氧化速度、CD谱、受体及抗体结合活性 ,以及对胰蛋白酶酶切敏感性的差别。结果表明 ,A7 B7二硫键缺失可导致胰岛素原α 螺旋明显减少以及对胰蛋白酶的酶切敏感性显著增加 ,其对胰岛素原结构的影响主要导致了受体结合活性的大幅度降低。突变体在体外重折叠 1h后巯基氧化速率较野生型明显减慢 ,但其最终折叠产率与野生型相当。由此提出一个胰岛素原折叠的可能途径 ,即A链链内二硫键最先形成 ,然后是两对链间二硫键。且A2 0 B19二硫键比A7 B7二硫键很可能先形成 ,在折叠中更重要。     

Insulin: a small protein with a long journey

Insulin is a hormone that is essential for regulating energy storage and glucose metabolism in the body. Insulin in liver, muscle, and fat tissues stimulates the cell to take up glucose from blood and store it as glycogen in liver and muscle. Failure of insulin control causes diabetes mellitus (DM). Insulin is the unique medicine to treat some forms of DM. The population of diabetics has dramatically increased over the past two decades, due to high absorption of carbohydrates (or fats and proteins), lack of physical exercise, and development of new diagnostic techniques. At present, the two largest developing countries (India and China) and the largest developed country (United States) represent the top three countries in terms of diabetic population. Insulin is a small protein, but contains almost all structural features typical of proteins: α-helix, β-sheet, β-turn, high order assembly, allosteric T®R-transition, and conformational changes in amyloidal fibrillation. More than ten years’ efforts on studying insulin disulfide intermediates by NMR have enabled us to decipher the whole picture of insulin folding coupled to disulfide pairing, especially at the initial stage that forms the nascent peptide. Two structural switches are also known to regulate insulin binding to receptors and progress has been made to identify the residues involved in binding. However, resolving the complex structure of insulin and its receptor remains a challenge in insulin research. Nevertheless, the accumulated knowledge of insulin structure has allowed us to specifically design a new ultra-stable and active single-chain insulin analog (SCI-57), and provides a novel way to design super-stable, fast-acting and cheaper insulin formulations for DM patients. Continuing this long journey of insulin study will benefit basic research in proteins and in pharmaceutical therapy.     

Insights into Coupled Folding and Binding Mechanisms from Kinetic Studies

Intrinsically disordered proteins (IDPs) are characterized by a lack of persistent structure. Since their identification more than a decade ago, many questions regarding their functional relevance and interaction mechanisms remain unanswered. Although most experiments have taken equilibrium and structural perspectives, fewer studies have investigated the kinetics of their interactions. Here we review and highlight the type of information that can be gained from kinetic studies. In particular, we show how kinetic studies of coupled folding and binding reactions, an important class of signaling event, are needed to determine mechanisms.     

Folding of a beta-sheet protein monitored by real-time NMR spectroscopy

At low ionic strength, apoplastocyanin forms an unfolded state under non-denaturing conditions. The refolding of this state is sufficiently slow to allow real-time NMR experiments to be performed. Folding of apoplastocyanin, initiated by the addition of salt and followed by real-time 2D 1H-15N heteronuclear single quantum coherence (HSQC) spectroscopy, is highly cooperative. A concomitant increase in the intensity of both sequential and long-range nuclear Overhauser effects (NOEs) between backbone amide protons in successive acquisitions of 1H-15N HSQC-NOESY-HSQC spectra provides the first direct observation of the development of structure-specific NOEs as a protein folds. Our results show that the local and long-range interactions in the native apoplastocyanin are formed simultaneously, consistent with highly cooperative formation of the native structure.     

Folding in vitro of bovine pancreatic trypsin inhibitor in the presence of proteins of the endoplasmic reticulum.

The rates of folding and disulfide bond formation in reduced BPTI were measured in vitro in the presence and absence of total protein from the endoplasmic reticulum. The rates were increased substantially by the endoplasmic reticulum proteins, but only to the extent expected from the known content and activity of protein-disulfide-isomerase. No effects of added ATP or Ca2+ were observed, even though protein-disulfide-isomerase binds Ca2+ tightly.     

Observing a late folding intermediate of Ubiquitin at atomic resolution by NMR

The study of intermediates in the protein folding pathway provides a wealth of information about the energy landscape. The intermediates also frequently initiate pathogenic fibril formations. While observing the intermediates is difficult due to their transient nature, extreme conditions can partially unfold the proteins and provide a glimpse of the intermediate states. Here, we observe the high resolution structure of a hydrophobic core mutant of Ubiquitin at an extreme acidic pH by nuclear magnetic resonance (NMR) spectroscopy. In the structure, the native secondary and tertiary structure is conserved for a major part of the protein. However, a long loop between the beta strands β3 and β5 is partially unfolded. The altered structure is supported by fluorescence data and the difference in free energies between the native state and the intermediate is reflected in the denaturant induced melting curves. The unfolded region includes amino acids that are critical for interaction with cofactors as well as for assembly of poly‐Ubiquitin chains. The structure at acidic pH resembles a late folding intermediate of Ubiquitin and indicates that upon stabilization of the protein's core, the long loop converges on the core in the final step of the folding process.     

GroEL Can Unfold Late Intermediates Populated on the Folding Pathways of Monellin

The modulation of the folding mechanism of the small protein single-chain monellin (MNEI) by the Escherichia coli chaperone GroEL has been studied. In the absence of the chaperone, the folding of monellin occurs via three parallel routes. When folding is initiated in the presence of a saturating concentration of GroEL, only 50-60% of monellin molecules fold completely. The remaining 40-50% of the monellin molecules remain bound to the GroEL and are released only upon addition of ATP. It is shown that the basic folding mechanism of monellin is not altered by the presence of GroEL, but that it occurs via only one of the three available routes when folding is initiated in the presence of saturating concentrations of GroEL. Two pathways become nonoperational because GroEL binds very tightly to early intermediates that populate these pathways in a manner that makes the GroEL-bound intermediates incompetent to fold. This accounts for the monellin molecules that remain GroEL-bound at the end of the folding reaction. The third pathway remains operational because the GroEL-bound early intermediate on this pathway is folding-competent, suggesting that this early intermediate binds to GroEL in a manner that is different from that of the binding of the early intermediates on the other two pathways. It appears, therefore, that the same protein can bind GroEL in more than one way. The modulation of the folding energy landscape of monellin by GroEL occurs because GroEL binds folding intermediates on parallel folding pathways, in different ways, and with different affinities. Moreover, when GroEL is added to refolding monellin at different times after commencement of refolding, the unfolding of two late kinetic intermediates on two of the three folding pathways can be observed. It appears that the unfolding of late folding intermediates is enabled by a thermodynamic coupling mechanism, wherein GroEL binds more tightly to an early intermediate than to a late intermediate on a folding pathway, with preferential binding energy being larger than the stability of the late intermediate. Hence, it is shown that GroEL can inadvertently and passively cause, through its ability to bind different folding intermediates differentially, the unfolding of late productive intermediates on folding pathways, and that its unfolding action is not restricted solely to misfolded or kinetically trapped intermediates.     

Uncoupling the Folding and Binding of an Intrinsically Disordered Protein

The relationship between helical stability and binding affinity was examined for the intrinsically disordered transactivation domain of the myeloblastosis oncoprotein, c-Myb, and its ordered binding partner, KIX. A series of c-Myb mutants was designed to either increase or decrease helical stability without changing the binding interface with KIX. This included a complimentary series of A, G, P, and V mutants at three non-interacting sites. We were able to use the glycine mutants as a reference state and show a strong correlation between binding affinity and helical stability. The intrinsic helicity of c-Myb is 21%, and helicity values of the mutants ranged from 8% to 28%. The c-Myb helix is divided into two conformationally distinct segments. The N-terminal segment, from K291–L301, has an average helicity greater than 60% and the C-terminal segment, from S304–L315, has an average helicity less than 10%. We observed different effects on binding when these two segments were mutated. Mutants in the N-terminal segment that increased helicity had no effect on the binding affinity to KIX, while helix destabilizing glycine and proline mutants reduced binding affinity by more than 1 kcal/mol. Mutants that either increased or decreased helical stability in the C-terminal segment had almost no effect on binding. However, several of the mutants reveal the presence of multiple conformations accessible in the bound state based on changes in enthalpy and linkage analysis of binding free energies. These results may explain the high level of sequence identity (> 90%), even at non-interacting sites, for c-Myb homologues.     

Folding transitions in calpain activator peptides studied by solution NMR spectroscopy

Calpastatin, the endogenous inhibitor of calpain, a cysteine protease in eukaryotic cells, is an intrinsically unstructured protein, which upon binding to the enzyme goes through a conformational change. Peptides calpA (SGKSGMDAALDDLIDTLGG) and calpC (SKPIGPDDAIDALSSDFTS), corresponding to the two conserved subdomains of calpastatin, are known to activate calpain and increase the Ca²⁺ sensitivity of the enzyme. Using solution NMR spectroscopy, here we show that calpA and calpC are disordered in water but assume an α‐helical conformation in 50% CD₃OH. The position and length of the helices are in agreement with those described in the literature for the bound state of the corresponding segments of calpastatin suggesting that the latter might be structurally primed for the interaction with its target. According to our data, the presence of Ca²⁺ induces a backbone rearrangement in the peptides, an effect that may contribute to setting the fine conformational balance required for the interaction of the peptides with calpain. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Single-Molecule Force Spectroscopy of Protein Folding

The use of force probes to induce unfolding and refolding of single molecules through the application of mechanical tension, known as single-molecule force spectroscopy (SMFS), has proven to be a powerful tool for studying the dynamics of protein folding. Here we provide an overview of what has been learned about protein folding using SMFS, from small, single-domain proteins to large, multi-domain proteins. We highlight the ability of SMFS to measure the energy landscapes underlying folding, to map complex pathways for native and non-native folding, to probe the mechanisms of chaperones that assist with native folding, to elucidate the effects of the ribosome on co-translational folding, and to monitor the folding of membrane proteins.     

Residual interactions in unfolded bile acid-binding protein by 19F NMR

The folding initiation mechanism of human bile acid-binding protein (BABP) has been examined by (19) F NMR. Equilibrium unfolding studies of BABP labeled with fluorine at all eight of its phenylalanine residues showed that at least two sites experience changes in solvent exposure at high denaturant concentrations. Peak assignments were made by site-specific 4FPhe incorporation. The resonances for proteins specifically labeled at Phe17, Phe47, and Phe63 showed changes in chemical shift at denaturant concentrations at which the remaining five phenylalanine residues appear to be fully solvent-exposed. Phe17 is a helical residue that was not expected to participate in a folding initiation site. Phe47 and Phe63 form part of a hydrophobic core region that may be conserved as a site for folding initiation in the intracellular lipid-binding protein family.