How the hydrophobic factor drives protein folding

How hydrophobicity (HY) drives protein folding is studied. The 1971 Nozaki–Tanford method of measuring HY is modified to use gases as solutes, not crystals, and this makes the method easy to use. Alkanes are found to be much more hydrophobic than rare gases, and the two different kinds of HY are termed intrinsic (rare gases) and extrinsic (alkanes). The HY values of rare gases are proportional to solvent-accessible surface area (ASA), whereas the HY values of alkanes depend on special hydration shells. Earlier work showed that hydration shells produce the hydration energetics of alkanes. Evidence is given here that the transfer energetics of alkanes to cyclohexane [Wolfenden R, Lewis CA, Jr, Yuan Y, Carter CW, Jr (2015) Proc Natl Acad Sci USA 112(24):7484–7488] measure the release of these shells. Alkane shells are stabilized importantly by van der Waals interactions between alkane carbon and water oxygen atoms. Thus, rare gases cannot form this type of shell. The very short (approximately picoseconds) lifetime of the van der Waals interaction probably explains why NMR efforts to detect alkane hydration shells have failed. The close similarity between the sizes of the opposing energetics for forming or releasing alkane shells confirms the presence of these shells on alkanes and supports Kauzmann''s 1959 mechanism of protein folding. A space-filling model is given for the hydration shells on linear alkanes. The model reproduces the n values of Jorgensen et al. [Jorgensen WL, Gao J, Ravimohan C (1985) J Phys Chem 89:3470–3473] for the number of waters in alkane hydration shells.When Kauzmann published his classic 1959 paper (1) on a new hydrophobic factor that drives protein folding, he gave examples from the literature (his table 3) of the energetics of transferring alkanes and aromatic hydrocarbons between various organic solvents and water. These examples show that the transfer free energy is favorable and sizable when a hydrocarbon solute is transferred from water to an organic solvent. Finding a favorable transfer free energy from water to an organic solvent prompted Kauzmann (1, 2) to suggest a protein-folding mechanism in which hydrocarbon side chains of the unfolded protein are driven to leave water and enter the folded protein interior because the interior is water free.Tanford (3) in 1962 then showed how the hydrophobic factor can be evaluated quantitatively [as the hydrophobicity (HY)] for amino acid side chains by using their solubilities in ethanol and water. Tanford (3) showed that the transfer free energy from water to ethanol can be found from the solubilities of the solute in water and ethanol, and he pointed out that the required solubility data are given in the book by Cohn and Edsall (4). By making free-energy calculations from these data, Tanford (3) in 1962 began the quantitative study of HY, and he argued that it is the key to understanding how proteins fold. In 1971, Nozaki and Tanford (5) gave the name HY to this type of analysis. The long-standing meaning of the term hydrophobic (water-hating) is that a hydrophobic solute is much more soluble in most organic solvents than in water (6). Nozaki and Tanford (5) used this meaning to develop a method of measuring HY values from the solute''s solubilities in water and ethanol, or other reference solvent. In 1971, they gave their measurements of HY values (5) for 12 amino acid side chains and for the peptide unit. Their 1971 HY values agree fairly well with the ones Tanford (3) gave in 1962, based on literature values for the solubilities.The 1971 Nozaki–Tanford paper (5) became an instant classic, and their method of measuring HY values was widely accepted. However, the 1971 Nozaki–Tanford method is difficult to use and was not used after 1971, not even by Tanford. To make the Nozaki–Tanford method of measuring HY values simpler to use, we modified the method to use gases rather than crystalline side chains as solutes. Some of the crystalline amino acids studied in 1971 by Nozaki and Tanford (5) were insoluble in both reference solvents, ethanol and dioxane, used by them. Thus, they had to make solubility measurements in mixtures of water and ethanol (or dioxane) to obtain measurable solubilities, and they needed to make difficult extrapolations to obtain solubility results for 100% ethanol or 100% dioxane. Using the modified method given here, with gases as solutes, there is no similar solubility problem.HY values are measured here for alkanes and rare gases. Alkanes are found to be much more hydrophobic than rare gases; in fact, there are two different kinds of HY. This result was expected from the proposal by Jorgensen et al. (7), who showed that van der Waals (vdW) interactions between alkane carbon and water oxygen atoms tether a fixed number (n) of water molecules to each of the seven alkanes they studied. Moreover, Jorgensen et al. (7) measured the Lennard–Jones potential of the C...O vdW interaction and found that it is quite strong. Jorgensen et al. (7) proposed that these tethered water molecules serve as Kauzmann''s (1, 2) hydration shells, which contribute to the solute''s HY. Thus, alkanes were expected to be more hydrophobic than rare gases because rare gases lack carbon atoms and cannot form these tethered water molecules.     

Identifying protein interactors in gonadotropin action

Systems biology integrates a variety of diverse approaches to the study of the cellular pathways comprised of protein networks. Following an initial ligand-receptor binding event, transduction of the signal is modified in a variety of ways via downstream protein interactions. Protein interactions can occur between two proteins or, alternatively, an interaction between two proteins can be facilitated by an adapter protein. Protein interactions can affect the spatial and temporal distribution of ligand-receptor complexes in cells, attenuating or prolonging signaling. With regard to gonadotropin receptors, protein interactions have been primarily studied in terms of desensitization and termination of signal transduction, or for their role in trafficking. The purpose of this review is to describe protein interactions that mediate gonadotropin receptor functions and to highlight some emerging interactions, as well as some of the caveats inherent in the attempt to uncover these pathways.     

Dynamic hydration shell restores Kauzmann's 1959 explanation of how the hydrophobic factor drives protein folding

Kauzmann''s explanation of how the hydrophobic factor drives protein folding is reexamined. His explanation said that hydrocarbon hydration shells are formed, possibly of clathrate water, and they explain why hydrocarbons have uniquely low solubilities in water. His explanation was not universally accepted because of skepticism about the clathrate hydration shell. A revised version is given here in which a dynamic hydration shell is formed by van der Waals (vdw) attraction, as proposed in 1985 by Jorgensen et al. [Jorgensen WL, Gao J, Ravimohan C (1985) J Phys Chem 89:3470–3473]. The vdw hydration shell is implicit in theories of hydrophobicity that contain the vdw interaction between hydrocarbon C and water O atoms. To test the vdw shell model against the known hydration energetics of alkanes, the energetics should be based on the Ben-Naim standard state (solute transfer between fixed positions in the gas and liquid phases). Then the energetics are proportional to n, the number of water molecules correlated with an alkane by vdw attraction, given by the simulations of Jorgensen et al. The energetics show that the decrease in entropy upon hydration is the root cause of hydrophobicity; it probably results from extensive ordering of water molecules in the vdw shell. The puzzle of how hydrophobic free energy can be proportional to nonpolar surface area when the free energy is unfavorable and the only known interaction (the vdw attraction) is favorable, is resolved by finding that the unfavorable free energy is produced by the vdw shell.When Kauzmann reviewed in 1959 (1) the possible sources of the free energy needed to drive protein folding, he found that that the known factors are not sufficient. He asked what the missing factor could be, and he ruled out peptide H-bonds because they do not provide enough free energy, based on Schellman''s (2) analysis of the problem in 1955. Then he discovered the previously unknown hydrophobic factor after observing that known DG values for transfer of hydrocarbons out of water into other solvents could supply the missing free energy. Then he needed to assume that the interior of a folded protein is water-free and the nonpolar side chains are buried inside the protein as folding occurs. In 1960 the 2-Å structure of myoglobin by Kendrew et al. (3) confirmed these predictions.     

Observation of ice-like water layers at an aqueous protein surface

We study the properties of water at the surface of an antifreeze protein with femtosecond surface sum frequency generation spectroscopy. We find clear evidence for the presence of ice-like water layers at the ice-binding site of the protein in aqueous solution at temperatures above the freezing point. Decreasing the temperature to the biological working temperature of the protein (0 °C to −2 °C) increases the amount of ice-like water, while a single point mutation in the ice-binding site is observed to completely disrupt the ice-like character and to eliminate antifreeze activity. Our observations indicate that not the protein itself but ordered ice-like water layers are responsible for the recognition and binding to ice.It is increasingly recognized that the conformational dynamics and the functioning of proteins are closely connected to the structure and dynamics of the surrounding water (1, 2). The idea of water being not just a passive spectator but an active player in dynamical processes in biosystems has gained ground both in experiment and theory (1–3). Especially, hydrophobic hydration is considered to play a key role in biological processes, ranging from protein folding to ligand binding (2, 4, 5). In the field of protein−solvent interactions, antifreeze proteins (AFPs) play an extraordinary role. These proteins must specifically recognize and bind nascent ice crystals within the vast excess of 55 M liquid water, and thus must be very sensitive to the structural differences between the two water phases. Despite this difficult molecular recognition problem, the success of AFPs as efficient protection against freezing is illustrated by a wide distribution of AFPs among psychrophilic organisms, such as insects, fish, plants, and bacteria living in freezing habitats (6–8). Each of these groups contains AFPs that have different evolutionary origins and a great diversity in structure. All AFPs are believed to function via an adsorption–inhibition mechanism in which the proteins adsorb to the surface of ice crystals and prevent their macroscopic growth (9). The ice recognition is performed at a specific side of the protein, known as the ice-binding site (IBS), which tends to be relatively flat and hydrophobic. The present work focuses on vibrational sum frequency generation spectroscopy (VSFG) of the AFP type three (AFP-III) from an Antarctic eelpout. AFP-III has been the subject of numerous experimental (10–13) and computational studies (14, 15), and these studies have identified the protein region that is responsible for the recognition of and interaction with the primary prism planes of ice, as shown in Fig. 1, Inset. The IBS of this globular protein of ∼7 kDa consists of a flat, relatively hydrophobic area where certain amino acid side chains are fixed in position and are believed to organize water molecules into a specific ice-like manner (11). Among those are the hydrophobic Gln9, Leu10, Ile13, Ala16, Leu19, Val20, Met21, Val51, and Gln44 as well as the hydrophilic Asn14, Thr15, and Thr18 (see Fig. S1) (10). The surface of liquids and solids can be probed with high selectivity with VSFG. In this technique, an infrared light pulse and a visible pulse are combined at the surface to generate light at their sum frequency. The generation is enhanced in case the infrared light is resonant with a molecular vibration at the surface. The technique is bulk forbidden due to symmetry, and thus highly surface specific. VSFG has been used to investigate the structure of interfacial water at various interfaces, including protein monolayers and organic molecules (16–19). AFP-III shows a strong propensity to localize at the hydrophobic water–air interface (20), thus offering the unique opportunity to study the properties of AFP-III’s hydrophobic IBS with vibrational sum frequency generation (VSFG).Open in a separate windowFig. 1.VSFG spectra of the water–air interface and an aqueous solution of a 93-μM aqueous AFP-III (pH 7.8) at room temperature. The VSFG spectrum of the water–air interface (black) consists of two broadbands at 3,200 cm⁻¹ and 3,450 cm⁻¹ assigned to hydrogen-bonded liquid water and a sharp peak at 3,700 cm⁻¹ assigned to dangling OH groups sticking out of the surface. The VSFG spectra of AFP-III (red) shows a single strong relatively narrow peak at 3,200 cm⁻¹ and spectral features associated with the CH vibrations of the protein at frequencies <3,000 cm⁻¹. Inset shows the 3D structure of AFP-III (1MSI) with the ice-binding surface highlighted in magenta.     

Gas-liquid transfer data used to analyze hydrophobic hydration and find the nature of the Kauzmann-Tanford hydrophobic factor

Hydrophobic free energy for protein folding is currently measured by liquid-liquid transfer, based on an analogy between the folding process and the transfer of a nonpolar solute from water into a reference solvent. The second part of the analogy (transfer into a nonaqueous solvent) is dubious and has been justified by arguing that transfer out of water probably contributes the major part of the free energy change. This assumption is wrong: transfer out of water contributes no more than half the total, often less. Liquid-liquid transfer of the solute from water to liquid alkane is written here as the sum of 2 gas-liquid transfers: (i) out of water into vapor, and (ii) from vapor into liquid alkane. Both gas-liquid transfers have known free energy values for several alkane solutes. The comparable values of the two different transfer reactions are explained by the values, determined in 1991 for three alkane solutes, of the cavity work and the solute-solvent interaction energy. The transfer free energy is the difference between the positive cavity work and the negative solute-solvent interaction energy. The interaction energy has similar values in water and liquid alkane that are intermediate in magnitude between the cavity work in water and in liquid alkane. These properties explain why the transfer free energy has comparable values (with opposite signs) in the two transfers. The current hydrophobic free energy is puzzling and poorly defined and needs a new definition and method of measurement.     

Protein-ice interaction of an antifreeze protein observed with solid-state NMR

NMR on frozen solutions is an ideal method to study fundamental questions of macromolecular hydration, because the hydration shell of many biomolecules does not freeze together with bulk solvent. In the present study, we present previously undescribed NMR methods to study the interactions of proteins with their hydration shell and the ice lattice in frozen solution. We applied these methods to compare solvent interaction of an ice-binding type III antifreeze protein (AFP III) and ubiquitin a non-ice-binding protein in frozen solution. We measured ¹H-¹H cross-saturation and cross-relaxation to provide evidence for a molecular contact surface between ice and AFP III at moderate freezing temperatures of -35 °C. This phenomenon is potentially unique for AFPs because ubiquitin shows no such cross relaxation or cross saturation with ice. On the other hand, we detected liquid hydration water and strong water–AFP III and water–ubiquitin cross peaks in frozen solution using relaxation filtered ²H and HETCOR spectra with additional ¹H-¹H mixing. These results are consistent with the idea that ubiquitin is surrounded by a hydration shell, which separates it from the bulk ice. For AFP III, the water cross peaks indicate that only a portion of its hydration shell (i.e., at the ice-binding surface) is in contact with the ice lattice. The rest of AFP III’s hydration shell behaves similarly to the hydration shell of non-ice-interacting proteins such as ubiquitin and does not freeze together with the bulk water.     

Structural diversity in the RGS domain and its interaction with heterotrimeric G protein alpha-subunits

Regulator of G protein signaling (RGS) proteins accelerate GTP hydrolysis by Galpha subunits and thus facilitate termination of signaling initiated by G protein-coupled receptors (GPCRs). RGS proteins hold great promise as disease intervention points, given their signature role as negative regulators of GPCRs-receptors to which the largest fraction of approved medications are currently directed. RGS proteins share a hallmark RGS domain that interacts most avidly with Galpha when in its transition state for GTP hydrolysis; by binding and stabilizing switch regions I and II of Galpha, RGS domain binding consequently accelerates Galpha-mediated GTP hydrolysis. The human genome encodes more than three dozen RGS domain-containing proteins with varied Galpha substrate specificities. To facilitate their exploitation as drug-discovery targets, we have taken a systematic structural biology approach toward cataloging the structural diversity present among RGS domains and identifying molecular determinants of their differential Galpha selectivities. Here, we determined 14 structures derived from NMR and x-ray crystallography of members of the R4, R7, R12, and RZ subfamilies of RGS proteins, including 10 uncomplexed RGS domains and 4 RGS domain/Galpha complexes. Heterogeneity observed in the structural architecture of the RGS domain, as well as in engagement of switch III and the all-helical domain of the Galpha substrate, suggests that unique structural determinants specific to particular RGS protein/Galpha pairings exist and could be used to achieve selective inhibition by small molecules.     

The evolutionary dynamics of the Saccharomyces cerevisiae protein interaction network after duplication

Gene duplication is an important mechanism in the evolution of protein interaction networks. Duplications are followed by the gain and loss of interactions, rewiring the network at some unknown rate. Because rewiring is likely to change the distribution of network motifs within the duplicated interaction set, it should be possible to study network rewiring by tracking the evolution of these motifs. We have developed a mathematical framework that, together with duplication data from comparative genomic and proteomic studies, allows us to infer the connectivity of the preduplication network and the changes in connectivity over time. We focused on the whole-genome duplication (WGD) event in Saccharomyces cerevisiae. The model allowed us to predict the frequency of intergene interaction before WGD and the post duplication probabilities of interaction gain and loss. We find that the predicted frequency of self-interactions in the preduplication network is significantly higher than that observed in today's network. This could suggest a structural difference between the modern and ancestral networks, preferential addition or retention of interactions between ohnologs, or selective pressure to preserve duplicates of self-interacting proteins.     

早老素1与热休克蛋白70同源蛋白羧基端相互作用蛋白

目的 探讨早老素(PSI)蛋白突变体在阿尔茨海默病(AD)发生中的作用和PSI蛋白的正常生理功能及分子伴侣蛋白热休克蛋白70同源蛋白羧基端相互作用蛋白(CHIP)的相互作用.方法 采用酵母双杂交系统筛选与PSI蛋白相互作用的蛋白.在构建pGBKT7-PS1-C203诱饵质粒和含全长CHIP的pACT2一CHIP质粒表达载体后,用p-半乳糖苷酶活性测定法检测两者的相互作用;然后转染哺乳动物细胞293T,用Co-IP和Western blot法测试其相瓦作用. 结果 获得了一个能与PSI蛋白相互作用的蛋白,即CHIP,并通过β-半乳糖甘酶活性测试、免疫共沉淀进一步证实了PSI和CHIP相互作用的特异性. 结论 CHIP既可以和分子伴侣蛋白相互作用,本身又具有泛素连接酶活性,可调控蛋白的折叠和降解,PSI与CHIP相互作用的证实,有助于阐明机体泛素-蛋白酶体系统对PSI的调控和进一步阐明AD的病理机制.     

肠易激综合征脑-肠交互作用模型结肠组织蛋白指纹图谱初探

目的 建立肠易激综合征(IBS)动物模型,利用基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)技术分析结肠组织差异蛋白质表达谱,为探索IBS发病机制提供线索.方法 雄性成年Wistar大鼠14只,随机分为模型组和正常组,每组7只.对模型组大鼠采用慢性轻度不可预见性应激联合急性束缚应激制作IBS慢急性联合应激大鼠模型,以行为学方法 评估模型.以MALDI-TOF-MS技术观察大鼠结肠蛋白质伞景,从整体上探索IBS这一功能性肠病有无差异表达蛋白.结果 (1)一般情况:模型组体重低于正常组[(298.88±18.61)g比(348.00±12.44)g,P<0.01];肠道动力:模型组大鼠制模后1 h的排便颗粒数明显多于正常组[(6.00±1.69)粒/1h比(1.14±0.69)粒1 h,P<0.01];行为检测:模型组与正常组相比,糖水消耗量显著减少[(13.63±1.69)ml/1 h比(19.00±3.06)ml/1 h,P<0.05];内脏敏感性:模型组在各个气囊容量下腹肌收缩次数均明显高于正常组(P<0.05).(2)MALDI-TOF-MS 鉴定结果 :模型组与正常组大鼠结肠组织有12个标志蛋白表达有明显差异,分为4类,分别与肠上皮细胞离子分泌、蛋白质合成、G蛋白系统、免疫有关;12种差异表达蛋白在模型组均高于正常组(P<0.05).结论 慢急性联合应激大鼠可部分模拟人类IBS脑-肠交互作用.差异蛋白质的检测为IBS发病机制及治疗靶点的选择提供了参考依据.     

乙型肝炎病毒X蛋白与细胞色素C氧化酶Ⅲ的特异性结合

目的 探讨HBV X蛋白与细胞色素c氧化酶Ⅲ(COXⅢ)之间的结合作用.方法 采用交合实验验证HBV X蛋白和COXⅢ在酵母细胞内的结合作用,利用离体结合实验证实两者在体外的结合作用,采用免疫共沉淀实验证实两者在哺乳动物细胞中的结合作用.结果 携带COXⅢ基因的酵母与携带X基因的酵母交合后发生反应,β-半乳糖苷酶(LacZ)活性检测菌落呈现蓝色,离体结合实验Western印迹中出现特异性结合活性反应条带,提示X蛋白和COXⅢ在体外存在直接相互作用,免疫共沉淀实验在相对分子质量为17 000处出现蛋白条带,提示在哺乳动物细胞水平仍能检测到X蛋白和COXⅢ的特异结合.结论 COXⅢ与X蛋白在原核细胞和哺乳动物细胞中均存在特异性结合,HBV可能通过此反应干扰线粒体呼吸链功能及能量代谢过程.     

ParA-like protein uses nonspecific chromosomal DNA binding to partition protein complexes

Recent data have shown that plasmid partitioning Par-like systems are used by some bacterial cells to control localization of protein complexes. Here we demonstrate that one of these homologs, PpfA, uses nonspecific chromosome binding to separate cytoplasmic clusters of chemotaxis proteins upon division. Using fluorescent microscopy and point mutations, we show dynamic chromosome binding and Walker-type ATPase activity are essential for cluster segregation. The N-terminal domain of a cytoplasmic chemoreceptor encoded next to ppfA is also required for segregation, probably functioning as a ParB analog to control PpfA ATPase activity. An orphan ParA involved in segregating protein clusters therefore uses a similar mechanism to plasmid-segregating ParA/B systems and requires a partner protein for function. Given the large number of genomes that encode orphan ParAs, this may be a common mechanism regulating segregation of proteins and protein complexes.     

丙型肝炎病毒核心蛋白结合蛋白激酶R的功能区域研究

目的 构建并表达丙型肝炎病毒(HCV)不同病毒株：癌中心株(BT)、癌旁珠(BNT)、HCV-J及BT不同截短片段谷胱甘肽(GST)-核心融合蛋白；寻找核心蛋白(Core)与蛋白激酶R(PKR)相互作用区域，探讨它们在HCV持续感染及肝细胞癌(HCC)发病机制中的作用。方法 用聚合酶链反应扩增不同片段HCV核心蛋白基因，并将7个不同的基因片段分别克隆到原核表达载体pGEX-4T-1，诱导表达并纯化表达蛋白，与两株细胞(HepG2和Huh-7)的PKR进行相互作用试验。结果7个不同片段Core在体外都得到相应表达，不同片段结合PKR的能力存在一定差异。BT、BNT、C191的Core N端1～172氨基酸(aa)3个片段均能与PKR发生直接结合，BT与PKR结合的区域在Core N端的1～58aa。结论 不同片段Core在原核细胞中获得较好的表达；Core／PKR相互作用，在HCV持续感染和HCC的发病机制中可能起重要作用。     

Host humoral immune response to Leishmania lipid-binding protein

SUMMARY We report on the use of Leishmania donovani lipid-binding proteins (LBPs) as antigens capable of being recognized by serum from immunocompetent patients from southern Spain suffering from visceral leishmaniasis and from Peruvian patients with localized cutaneous leishmaniasis caused by Leishmania braziliensis. The absorbance found by immunoenzymatic techniques gave significantly different results for the serum samples from patients with and without leishmaniasis. Specificity by ELISA testing was 93.2% and sensibility 100%. Dot blots from human patient serum samples or naturally infected dogs from Spain gave similarly significant results. All the human serum samples from individuals with visceral leishmaniasis and the Leishmania-positive canine samples recognized two bands, with molecular weights of 8 and 57 kDa. The serum from individuals with cutaneous leishmaniasis caused by L. braziliensis recognized an additional band of 16 kDa. We discuss the role of Leishmania FABP and compare the immunological reactions found with serum samples from other protozoan infections such as toxoplasma and Chagas as well as bacterial infections such as tuberculosis and syphilis.     

蛋白电泳凝胶放置时间对蛋白丰度的影响分析

目的利用高分辨力的双向电泳技术检测蛋白电泳后凝胶中的蛋白含量随时间推移的损失情况。方法用双向电泳蛋白裂解液提取鼠疫EV菌株(无毒疫苗株)全菌蛋白并定量。取800μg EV蛋白,平行进行两块双向电泳凝胶电泳,电泳结束后一块凝胶立刻固定并染色,另一块凝胶4℃放置8h后固定并染色,使用ImageMaster 2DElite软件比较分析两块凝胶蛋白点数量及蛋白丰度。结果相比直接固定染色的凝胶,8h后固定染色的凝胶中蛋白点扩散丢失严重,比电泳后直接固定凝胶少346个蛋白点,且部分相邻蛋白点发生融合,含量在20~350ng的低丰度蛋白65.9%因蛋白扩散丢失。结论蛋白凝胶放置8h后大量低丰度蛋白由于扩散会丢失,并直接影响后续免疫印迹、质谱鉴定等实验结果的可靠性。     

HIV-1 Tat protein and endothelium: from protein/cell interaction to AIDS-associated pathologies

Tat protein, the transactivating factor of the human immunodeficiency virus type 1 (HIV-1), is a small cationic polypeptide that can be released from HIV-1 infected cells. Extracellular Tat elicits different biological responses in several types of target cells, including endothelial cells (ECs). In the present paper, we will review the various aspects from the laboratory bench to the bedside that characterize the tight relationship that exists between HIV-1 Tat and the endothelium. Tat interacts with at least three different types of receptors present on the surface of ECs. This leads to the activation of several signal transduction pathways and triggers various biological responses in the endothelium. The bioavailability, cell interaction, intracellular signaling, and biological activity of Tat are tightly regulated by components of the extracellular matrix and circulating molecules. Thus, Tat is at the center of a complex network of interactions that occur at the surface of ECs and that greatly affect the functions of the endothelium, possibly resulting in some of the pathological processes that occur in AIDS patients.     

Large-scale modulation of thermodynamic protein folding barriers linked to electrostatics

Protein folding barriers, which range from zero to the tens of RT that result in classical two-state kinetics, are primarily determined by protein size and structural topology [Plaxco KW, Simons KT, Baker D (1998) J Mol Biol 277:985–994]. Here, we investigate the thermodynamic folding barriers of two relatively large proteins of the same size and topology: bovine α-lactalbumin (BLA) and hen-egg-white lysozyme (HEWL). From the analysis of differential scanning calorimetry experiments with the variable-barrier model [Muñoz V, Sanchez-Ruiz JM (2004) Proc Natl Acad Sci USA 101:17646–17651] we obtain a high barrier for HEWL and a marginal folding barrier for BLA. These results demonstrate a remarkable tuning range of at least 30 kJ/mol (i.e., five to six orders of magnitude in population) within a unique protein scaffold. Experimental and theoretical analyses on these proteins indicate that the surprisingly small thermodynamic folding barrier of BLA arises from the stabilization of partially unfolded conformations by electrostatic interactions. Interestingly, there is clear reciprocity between the barrier height and the biological function of the two proteins, suggesting that the marginal barrier of BLA is a product of natural selection. Electrostatic surface interactions thus emerge as a mechanism for the modulation of folding barriers in response to special functional requirements within a given structural fold.     

丙型肝炎病毒非结构蛋白4A与钙离子信号调节亲环素配体的相互作用

目的 应用酵母双杂交技术及免疫共沉淀技术证实HCV非结构蛋白4A（HCV NS4A）与钙离子信号调节亲环素配体（CAML）的相互作用。方法 对CAML编码基因进行克隆，并构建其酵母表达载体，在酵母细胞中与HCV NS4A进行回交验证后，构建HCV NS4A及CAML真核表达载体，转染293细胞，行免疫共沉淀实验及Western印迹检测。结果 成功克隆CAML基因，并构建CAML的酵母表达载体，在酵母细胞中回交验证HCV NS4A与CAML的相互作用，构建HCV NS4A及CAML的真核细胞表达载体，并利用免疫共沉淀技术验证了两者的相互作用。结论 CAML与HCV NS4A的相互作用可能对HCV感染细胞的钙离子浓度、细胞凋亡等生物过程产生影响，从而在HCV感染慢性化过程中发挥作用。