Compositional and Structural Features Related to Thermal Stability in the Archaea SRP19 and SRP54 Signal Recognition Particle Proteins

To survive at high temperature, thermophile organisms must adapt their biomolecules. In both nucleic acids and proteins, this adaptation involves a vast array of compositional and structural modifications. The archaea stand out as the only group of organisms that have species capable of growing at temperatures ranging from 0 to 110°C. In this study, we have used the archaea genome datasets to identify molecular trends related to thermal adaptation in the protein components (SRP19 and SRP54) of the signal recognition particle (SRP). Using comparative genomics and secondary structure homology modeling we have detected significant differences in the amino acids composition and distribution between the SRP proteins of thermophile and mesophile archaea. These include: a significant increase in the thermophile SRP proteins of the frequency of charged amino acids able to participate in electrostatic interactions which contribute to stabilize proteins; decreased content of both thermolabile and small/tiny amino acids which usually contribute to protein flexibility; and a significant increase in aliphatic and aromatic amino acids providing good covering and masking to produce hydrophobic pockets involved in stabilizing protein structure. Moreover, a detailed analysis of the four structural and functional domains of the SRP54 indicates a particularly robust correlation between the compositional properties of the M domain and the optimal growth temperature (OGT) of the archaea. The analysis of the bacterial SRP54(Ffh) shows similar adaptations to the OGT. Thus, natural selection has adapted the SRP proteins to the OGT of the archaea and bacteria species by modifying both, their amino acids composition and distribution.     

Hydrophobicity Density Profiles to Predict Thermal Stability Enhancement in Proteins

A hydrophobicity density is defined for a protein through its hydrophobicity tensor (similar to the inertia tensor), by using the Eisenberg hydrophobicity scale of the hydrophobic amino acids of a protein. This allows calculation of the radii of the corresponding hydrophobic ellipsoid of a protein and thus subsequently of its hydrophobic density. A hydrophobicity density profile is then obtained by simulating point mutations of each amino acid of a protein either to a high hydrophobicity value or to zero hydrophobicity. It is found that an increase in the hydrophobic density of the protein correlates with an increase of its mid-point transition temperature. From this profile it is possible to determine the amino acids or domain stretches in a protein that are most amenable to mutation in order to increase the thermal stability. The model is tested to predict the thermostabilisation effects of two mutations in a β-glucanase: M29G and M29F. This model is compared with other hydrophobicity-related profiles described by other authors.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Effect of Adsorbed Proteins on the Rheological Properties of Erythrocytes

Impedance spectroscopy in the radio-frequency band was used to probe the adsorptive properties of red blood cells. Fluidity assays of concentrated erythrocyte suspensions revealed a close relationship between the protein adsorption on erythrocyte membranes and the extent of cell interaction in the flow. Patients with impaired peripheral circulation displayed an increase in adsorption of high-molecular-weight proteins and a dramatic decrease in fluidity of erythrocyte suspensions. Hence, adsorption was assumed to contribute to rheological disorders.     

Probing Stability and Dynamics of Proteins by Protease Digestion I: Comparison of Protease Susceptibility and Thermal Stability of Cytochromes c

Abstract

Protease susceptibility of homologous proteins in their native conformations was studied. This work aims to establish a broad and quantitative basis for the utilization of protease digestion to analyze the local stability of native proteins. Using high-performance liquid chromatography (HPLC) the time course of the proteolytic degradation of intact proteins was quantitatively traced. Rapid separation of peptide fragments with HPLC made possible the elucidation of sequential digestion originating from the cleavage at a very few sites which are locally unstable in the protein structure. Using four serine proteases, chymotrypsin, trypsin, elastase and subtilisin BPN', we found some common trends in proteolysis for a group of proteins of the cytochrome c family. By comparing of the proteolysis and thermal denaturation with ten homologous cytochromes c extracted from horse, beef, Candida krusei, Saccharomyces cerevisiae, chicken, tuna, pigeon, rabbit, dog and rat, protease susceptibility was related to locally unfolding states intrinsic to the native conformation.     

蛋白质的结构转换

许多不相关的蛋白质含有相同的短肽序列却形成不同的空间构象. 结构转换广泛存在于蛋白质折叠和功能过程中, 具有重要的生物学意义. 综述了Serpin和EF-Tu的失活、血细胞凝集素的激活、蛋白酶成熟、亚基装配和蛋白质淀粉样化等过程中肽链同源肽段的结构转换模式, 并讨论了它在理解蛋白质折叠机理和“构象病”病因中的应用.     

Structural Proteins of Reoviruses

Polyacrylamide gel electrophoresis of the solubilized proteins from the three serotypes of reovirus revealed that each contained three major and four minor components. Subviral particles were prepared by brief treatment of complete virions with urea. Electron microscopy, density-gradient centrifugation, and chemical analyses of these particles indicated that their outer capsid structure had been selectively removed. They contained only two proteins, but their ribonucleic acid composition was similar to that of complete virions. The subviral particles were not infectious.     

Structural Maintenance of Chromosome (SMC) Proteins Link Microtubule Stability to Genome Integrity

Structural maintenance of chromosome (SMC) proteins are key organizers of chromosome architecture and are essential for genome integrity. They act by binding to chromatin and connecting distinct parts of chromosomes together. Interestingly, their potential role in providing connections between chromatin and the mitotic spindle has not been explored. Here, we show that yeast SMC proteins bind directly to microtubules and can provide a functional link between microtubules and DNA. We mapped the microtubule-binding region of Smc5 and generated a mutant with impaired microtubule binding activity. This mutant is viable in yeast but exhibited a cold-specific conditional lethality associated with mitotic arrest, aberrant spindle structures, and chromosome segregation defects. In an in vitro reconstitution assay, this Smc5 mutant also showed a compromised ability to protect microtubules from cold-induced depolymerization. Collectively, these findings demonstrate that SMC proteins can bind to and stabilize microtubules and that SMC-microtubule interactions are essential to establish a robust system to maintain genome integrity.     

Structural Aspects of Hydroxyproline-Containing Proteins

Abstract

The occurrence of hydroxyproline (Hyp) in collagen, Clq and acetylcholinesterase (AChE) raises important questions concerning the role of this unusual imino acid in the structure and function of these proteins. Available data on collagen indicate that Hyp is necessary for the normal secretion of the protein after its synthesis and for the integrity of the triple-helical conformation. Studies from our laboratory have dealt with the structural aspects of the posttranslational conversion of proline to hydroxyproline in collagen mediated by prolyl hydroxylase. We proposed that the β-turn conformation at the Pro-Gly segments in the nascent procollagen molecule are the sites of the enzymatic hydroxylation and that this conformation changes over to the collagen-like helix as a result of the hydroxylation process. Recently, we have provided additional experimental support to our proposal by a) synthesizing specific β-turn oligopeptides containing the Pro-Gly as well as Pro-Ala and Pro-DAla sequences and showing that these act as inhibitors of the enzymatic hydroxylation of a synthetic substrate and b) demonstrating, by circular dichroism spectroscopy, the occurrence of a conformational change leading to the triple-helix as a direct consequence of proline hydroxylation in a non-helical polypeptide substrate. We have also observed that the acquisition of hydroxylation results in a significant enhancement of the rate of folding of the polypeptide chain from the unfolded to the triple-helical conformation. We believe that our observations on proline hydroxylation in collagen should also be applicable to Clq and acetylcholineesterase both of which share the general structural and functional properties of collagen in their “tail” regions. Using the techniques employed in collagen studies, one should be able to assess the role of hydroxyproline in the folding, structural stabilities and functions of Clq and AChE. This would also involve the study of the unhydroxylated and hydroxylated precursors of these proteins which may share common structural features with their collagen counterparts. Finally, a systematic study of hydroxyproline-containing peptides and polypeptides has been initiated by us so as to understand the exact manner in which Hyp participates in the formation and stability of the triple-helical conformation in the proteins in which it occurs.     

Structural Determinants for Improved Stability of Designed Ankyrin Repeat Proteins with a Redesigned C-Capping Module

Designed ankyrin repeat proteins (DARPins) that specifically bind to almost any target can be obtained by ribosome display or phage display from combinatorial libraries. Although DARPins are already very stable molecules, molecular dynamics simulations, equilibrium denaturation experiments, structural studies, and recent NMR experiments suggested that the unfolding of the original C-terminal capping repeat (C-cap), taken from a natural ankyrin repeat protein, limits the stability of the initial DARPin design. Several point mutations had been introduced to optimize the C-cap and were shown to indeed further increase the stability of DARPins. We now determined crystal structures of DARPins with one or three full-consensus internal repeats (NI₁C or NI₃C) between an N-terminal capping repeat and mutants of the C-cap. An NI₁C mutant, in which the C-cap was only extended by three additional helix-forming residues, showed no structural change but reduced B-factors in the C-cap. An NI₃C C-cap mutant carrying five additional mutations in the interface to the preceding repeat, previously designed by using the consensus sequence as a guide, showed a rigid-body movement of the C-cap towards the internal repeat. This movement results in an increased buried surface area and a superior surface complementarity and explains the improved stability in equilibrium unfolding, compared to the original C-cap. A C-cap mutant with three additional mutations introducing suitably spaced charged residues did not show formation of salt bridges, explaining why its stability was not increased further. These structural studies underline the importance of repeat coupling for stability and help in the further design of this protein family.     

Structural Proteins of Rabies Virus

Purified rabies virions, unlabeled or labeled with radioactive amino acids or d-glucosamine, were dissociated into their polypeptides by treatment with sodium dodecyl sulfate in a reducing environment and fractionated by electroiphoresis in sodium dodecyl sulfate-containing polyacrylamide gel. The molecular weights of individual polypeptides were estimated by comparison of their rate of migration with that of protein markers of known molecular weight. Purified viral nucleocapsid and a mixture of envelope components, isolated from virions disrupted by sodium deoxycholate, were analyzed by the same procedure. The number of molecules per virion of each polypeptide was estimated from the proportions of the separated components, the known molecular weight of the viral ribonucleic acid, and the chemical composition of the nucleocapsid. The protein moiety of the nucleocapsid particle was estimated to consist of 1,713 molecules of a major polypeptide (molecular weight, 62,000 daltons) and 76 molecules of a minor polypeptide (molecular weight, 55,000 daltons). In addition to 1,783 molecules of a glycoprotein component (molecular weight, 80,000 daltons), the viral envelope contains 789 and 1,661 molecules, respectively, of two other polypeptides (molecular weight, 40,000 and 25,000 daltons).     

Structural Proteins of Pichinde Virus

Pichinde virus, a member of the arenovirus group, was found to have four polypeptides by polyacrylamide gel electrophoresis. Two components, V(I) and V(II), had molecular weights of about 72,000, whereas V(III) had a molecular weight of 34,000. A minor component, V(IV), had a molecular weight of about 12,000. Glucosamine was incorporated into V(II) and V(III), suggesting that these components were glycopeptides whereas V(I) and V(IV) were polypeptides. Treatment of the virus with Nonidet P-40 removed V(III), but V(I) and V(II) remained associated with the virus nucleic acid. This suggests a functional role of a ribonucleoprotein for V(I) and an envelope glycoprotein for V(III). V(II), the major glycopeptide, could function both as a membrane component and as a nucleoprotein.     

Structural Proteins of Adenovirus-Associated Viruses

The structural proteins of adenovirus-associated virus (AAV) types 1, 2, and 3 were analyzed by acrylamide gel electrophoresis. In each case, one major protein (C) and two minor proteins (A and B) were identified. Component C had an estimated molecular weight of 62,000 daltons, and the molecular weights of components A and B were found to be 87,000 and 73,000 daltons, respectively. Coelectrophoresis of adenovirus and AAV proteins revealed an overlap only between the adenovirus fiber-penton component and the AAV C polypeptide. Among AAV serotypes, homologous components were electrophoretically identical, except that the C component of AAV-2 was of slightly lower molecular weight than the C components of AAV-1 and AAV-3. The relative incorporation of (14)C-arginine and (14)C-mixed amino acids into the three polypeptides of AAV-2 was similar, indicating an absence of an arginine-rich component. In addition, AAV-2 was found to have a substantially lower arginine content than helper adenoviruses.     

吸附精制百日咳菌苗、白喉、破伤风类毒素混合制剂稳定性的研究

将连续生产并已申报人体接种反应观察的三批吸附精制百日咳菌苗、白喉、破伤风类毒素混合制剂,分别于４～８℃、１８～２２℃、３７℃贮存半年、１年、１年半和３０天,然后以小鼠法或豚鼠法对该制剂进行安全稳定性、效力稳定性以及毒性逆转等测试。其毒力安全稳定性试验表明,三批混合制剂中的精制百日咳菌苗在４～８℃贮半年、１年、１年半后、它们的ＢＷＤＵ／ｍｌ、ＬＰＵ／ｍｌ、ＨＳＵ／ｍｌ均无显著性差异（Ｐ＞０．０５）;白喉及破伤风类毒素在４～８℃贮存半年,１年、１年半以及１８～２２℃贮存半年和３７℃贮存３０天后,三批测试组的豚鼠均健存且体重增加。在效力稳定性试验中,４～８℃贮存１年和１年半后精制百日咳菌苗的效力与贮存前（分别为２６．７３ＩＵ／ｍｌ、２１．０２ＩＵ／ｍｌ、１９．１７ＩＵ／ｍｌ）无显著性差异（ｔ＝０．１１４;Ｐ＞０．０５）;白喉及破伤风类毒素４～８℃贮存长达１年半后,效力仍在２～３．２ＩＵ／ｍｌ,动物保护率仍可达８０～１００％。三批混合制剂在３７℃贮存３０天后,通过Ｓｃｈｉｃｋ毒性试验及测定ＢＷＤＵ／ｍｌ、ＬＰＵ／ｍｌ、ＨＳＵ／ｍｌ各指标显示,精制百日咳菌苗、白喉和破伤风类毒素无毒性逆转。说明该吸附精制百、白、破混合     

Structural Proteins and Subunits of Rubella Virus

Polyacrylamide gel electrophoresis of purified rubella virus revealed two distinct structural proteins VP1 and VP3, which had molecular weights of 62,500 and 35,000, respectively. In addition, a broad variable peak, designated VP2, with a molecular weight of about 47,500, was seen. Sucrose gradient analysis of virus disrupted by neutral detergents separated a labile 150S ribonucleoprotein, containing 40S ribonucleic acid and VP3, from the envelope fraction containing VP1 and VP2. VP1 and particularly VP2 were labeled with glucosamine and are thus glycoproteins. Labeling the polypeptides with different amino acids indicated that VP3, the "core" protein, is relatively rich in arginine but not in lysine. The size of the two main polypeptides, VP1 and VP3, corresponds to those of group A arboviruses.     

Structural and Nonstructural Proteins of an Arbovirus

Purified Semliki Forest virus (SFV) contains three structural proteins while its core (nucleocapsid) contains two of these proteins. To identify all of the proteins synthesized under virus direction, cells were infected with SFV in the presence of actinomycin D and guanidine. Cell protein synthesis was markedly and irreversibly inhibited under these conditions; virus growth was reversibly inhibited by guanidine and began when the cells were washed to remove the guanidine. When cells were treated with guanidine for 4 hr after virus infection and then were washed, five major proteins were produced early in infection. Three of these proteins corresponded to virus structural proteins. None of these five proteins was a major protein of uninfected cells or of virus-infected cells which had been incubated with partially purified interferon before infection. Late in infection, three major proteins, the virus structural proteins, were produced.     

解读AGO蛋白结构及其功能

RNA沉默是由小RNA特异向导和RNA诱导的沉默复合物（RISC）切割或者抑制靶标mRNA翻译的一种调控系统. 作为RISC的核心成分,AGO蛋白(argonaute proteins)由N末端、PAZ、MID和PIWI 4个结构域组成. PAZ区能非序列特异性识别结合双链小RNA 3′末端悬垂的2个核苷酸,MID与PIWI界面处的“保守口袋”识别结合小RNA 5′端第1位核苷酸,PIWI区具有切割mRNA的催化中心. 根据系统进化学分析,AGO蛋白家族分为3个组：AGO like、PIWI-like和GROUP3. 拟南芥共编码10种AGO蛋白.目前已经证实,具有切割活性的为AtAGO1、AtAGO4和AtAGO7,三者参与的小RNA通路也已得到确认. 在拟南芥10种AGO蛋白中,AtAGO1与AtAGO10、AtAGO1与AtAGO7、AtAGO4与AtAGO6存在功能上的部分冗余.     

Proteins of Yaba Monkey Tumor Virus I. Structural Proteins

Yaba virus proteins were characterized by polyacrylamide gel electrophoresis. Electrophoresis of Yaba virion (proteins) dissociated by sodium dodecyl sulfate and 2-mercaptoethanol in continuous and discontinuous buffer systems yielded 37 polypeptide species by staining and by counting bands of radioactively labeled polypeptides. The molecular weights of the viral polypeptide species were found to range from 10,000 to 220,000 by comparing the relative distance of migration of viral proteins with proteins of known molecular weights. Two polypeptides were removed from purified virions by nonionic detergent nonidet P -40 treatment, and the amount of one polypeptide was reduced. Purified cores yielded 21 polypeptide species, none of which was labeled with radioactive glucosamine.     

Differential Thermal Analysis of Milk Proteins

Differential thermal analysis (DTA) was used for study of milk protein denaturation. Protein solutions produced an endothermic peak of characteristic shape and temperature of peak minimum. The peak minimum is considered the coagulation temperature of the protein.

The influence of pH and additives such as sugars and NaCl was clearly observed on the thermograms of β-lactoglobulin solution. Addition of κ-casein to β-lactoglobulin solution showed an inhibitory effect on the heat coagulation.

Solid proteins produced two-stage exothermic peaks between 200°C and 400°C.

DTA was a useful method in the study of heat denaturation and degradation of protein.     

Structural Proteins of Simian Virus 40

Sodium dodecyl sulfate acrylamide gel electrophoresis of the solubilized proteins from purified simian virus 40 (SV40) virions revealed two major and two minor structural polypeptide components. The major components which comprise over 75% of the total virion were shown to be the capsid proteins by immunological and isoelectric focusing fractionation analysis. These two polypeptides have estimated molecular weights of 45,000 daltons as determined by gel electrophoresis. One of the two minor components was identified as the nucleocapsid protein and has an approximate molecular weight of 16,000. The other unidentified minor component has an average molecular weight of 29,000.