Structural Basis for Conformational Dynamics of GTP-bound Ras Protein

Ras family small GTPases assume two interconverting conformations, “inactive” state 1 and “active” state 2, in their GTP-bound forms. Here, to clarify the mechanism of state transition, we have carried out x-ray crystal structure analyses of a series of mutant H-Ras and M-Ras in complex with guanosine 5′-(β,γ-imido)triphosphate (GppNHp), representing various intermediate states of the transition. Crystallization of H-RasT35S-GppNHp enables us to solve the first complete tertiary structure of H-Ras state 1 possessing two surface pockets unseen in the state 2 or H-Ras-GDP structure. Moreover, determination of the two distinct crystal structures of H-RasT35S-GppNHp, showing prominent polysterism in the switch I and switch II regions, reveals a pivotal role of the guanine nucleotide-mediated interaction between the two switch regions and its rearrangement by a nucleotide positional change in the state 2 to state 1 transition. Furthermore, the ³¹P NMR spectra and crystal structures of the GppNHp-bound forms of M-Ras mutants, carrying various H-Ras-type amino acid substitutions, also reveal the existence of a surface pocket in state 1 and support a similar mechanism based on the nucleotide-mediated interaction and its rearrangement in the state 1 to state 2 transition. Intriguingly, the conformational changes accompanying the state transition mimic those that occurred upon GDP/GTP exchange, indicating a common mechanistic basis inherent in the high flexibility of the switch regions. Collectively, these results clarify the structural features distinguishing the two states and provide new insights into the molecular basis for the state transition of Ras protein.     

Protein Sliding along DNA: Dynamics and Structural Characterization

Efficient search of DNA by proteins is fundamental to the control of cellular regulatory processes. It is currently believed that protein sliding, hopping, and transfer between adjacent DNA segments, during which the protein nonspecifically interacts with DNA, are central to the speed of their specific recognition. In this study, we focused on the structural and dynamic features of proteins when they scan the DNA. Using a simple computational model that represents protein-DNA interactions by electrostatic forces, we identified that the protein makes use of identical binding interfaces for both nonspecific and specific DNA interactions. Accordingly, in its one-dimensional diffusion along the DNA, the protein is bound at the major groove and performs a helical motion, which is stochastic and driven by thermal diffusion. A microscopic structural insight into sliding from our model, which is governed by electrostatic forces, corroborates previous experimental studies suggesting that the active site of some regulatory proteins continually faces the interior of the DNA groove while sliding along sugar-phosphate rails. The diffusion coefficient of spiral motion along the major groove of the DNA is not affected by salt concentration, but the efficiency of the search can be significantly enhanced by increasing salt concentration due to a larger number of hopping events. We found that the most efficient search comprises ∼ 20% sliding along the DNA and ∼ 80% hopping and three-dimensional diffusion. The presented model that captures various experimental features of facilitated diffusion has the potency to address other questions regarding the nature of DNA search, such as the sliding characteristics of oligomeric and multidomain DNA-binding proteins that are ubiquitous in the cell.     

The Influence of Secondary Processing on the Structural Relaxation Dynamics of Fluticasone Propionate

This study investigated the structural relaxation of micronized fluticasone propionate (FP) under different lagering conditions and its influence on aerodynamic particle size distribution (APSD) of binary and tertiary carrier-based dry powder inhaler (DPI) formulations. Micronized FP was lagered under low humidity (LH 25 C, 33% RH [relative humidity]), high humidity (HH 25°C, 75% RH) for 30, 60, and 90 days, respectively, and high temperature (HT 60°C, 44% RH) for 14 days. Physicochemical, surface interfacial properties via cohesive-adhesive balance (CAB) measurements and amorphous disorder levels of the FP samples were characterized. Particle size, surface area, and rugosity suggested minimal morphological changes of the lagered FP samples, with the exception of the 90-day HH (HH90) sample. HH90 FP samples appeared to undergo surface reconstruction with a reduction in surface rugosity. LH and HH lagering reduced the levels of amorphous content over 90-day exposure, which influenced the CAB measurements with lactose monohydrate and salmeterol xinafoate (SX). CAB analysis suggested that LH and HH lagering led to different interfacial interactions with lactose monohydrate but an increasing adhesive affinity with SX. HT lagering led to no detectable levels of the amorphous disorder, resulting in an increase in the adhesive interaction with lactose monohydrate. APSD analysis suggested that the fine particle mass of FP and SX was affected by the lagering of the FP. In conclusion, environmental conditions during the lagering of FP may have a profound effect on physicochemical and interfacial properties as well as product performance of binary and tertiary carrier-based DPI formulations.

Electronic supplementary material

The online version of this article (doi:10.1208/s12249-014-0222-8) contains supplementary material, which is available to authorized users.KEY WORDS: cohesive-adhesive balance, laagering, mechanical activation, particle adhesion, process-induced structural disorder     

Role of Structural Dynamics at the Receptor G Protein Interface for Signal Transduction

GPCRs catalyze GDP/GTP exchange in the α-subunit of heterotrimeric G proteins (Gαßγ) through displacement of the Gα C-terminal α5 helix, which directly connects the interface of the active receptor (R*) to the nucleotide binding pocket of G. Hydrogen–deuterium exchange mass spectrometry and kinetic analysis of R* catalysed G protein activation have suggested that displacement of α5 starts from an intermediate GDP bound complex (R*•G^GDP). To elucidate the structural basis of receptor-catalysed displacement of α5, we modelled the structure of R*•G^GDP. A flexible docking protocol yielded an intermediate R*•G^GDP complex, with a similar overall arrangement as in the X-ray structure of the nucleotide free complex (R*•G^empty), however with the α5 C-terminus (GαCT) forming different polar contacts with R*. Starting molecular dynamics simulations of GαCT bound to R* in the intermediate position, we observe a screw-like motion, which restores the specific interactions of α5 with R* in R*•G^empty. The observed rotation of α5 by 60° is in line with experimental data. Reformation of hydrogen bonds, water expulsion and formation of hydrophobic interactions are driving forces of the α5 displacement. We conclude that the identified interactions between R* and G protein define a structural framework in which the α5 displacement promotes direct transmission of the signal from R* to the GDP binding pocket.     

Elucidating in Vivo Structural Dynamics in Integral Membrane Protein by Hydroxyl Radical Footprinting

We describe here a novel footprinting technique to probe the in vivo structural dynamics of membrane protein. This method utilized in situ generation of hydroxyl radicals to oxidize and covalently modify biomolecules on living Escherichia coli cell surface. After enriching and purifying the membrane proteome, the modified amino acid residues of the protein were identified with tandem mass spectrometry to map the solvent-accessible surface of the protein that will form the footprint of in vivo structure of the protein. Of about 100 outer membrane proteins identified, we investigated the structure details of a typical β-barrel structure, the porin OmpF. We found that six modified tryptic peptides of OmpF were reproducibly detected with 19 amino acids modified under the physiological condition. The modified amino acid residues were widely distributed in the external loop area, β-strands, and periplasmic turning area, and all of them were validated as solvent-accessible according to the crystallography data. We further extended this method to study the dynamics of the voltage gating of OmpF in vivo using mimic changes of physiological circumstance either by pH or by ionic strength. Our data showed the voltage gating of porin OmpF in vivo for the first time and supported the proposed mechanism that the local electrostatic field changes in the eyelet region may alter the porin channels to switch. Thus, this novel method can be a potentially efficient method to study the structural dynamics of the membrane proteins of a living cell.One of the most challenging problems in biological sciences is the correlation of the dynamic three-dimensional (3D)¹ structural changes in membrane proteins to their biological functions. Comprising about 30% of the human proteome, membrane proteins are critical mediators of material and information transfer between cells and their environment and are targets for many growth factors and pharmacologically active compounds. Signals from binding of ligands or drugs to receptors are transduced through conformational changes in the receptors. Therefore, understanding the dynamic conformational changes in the structure of membrane proteins is essential to the understanding of many biological processes and has important implications in human health.Although some methodologies including NMR and x-ray have been developed to study protein structures in atomic resolution, the determination of the structures of integral membrane proteins (IMPs) remains one of the most challenging problems in biological sciences (1). IMPs are usually insoluble low abundance proteins for which expression, purification, and crystallization are generally too inefficient to generate sufficient materials for conventional structural analysis techniques such as NMR and x-ray. Additionally, IMPs in living cells are constantly interacting with different molecules depending on the external environment and biological states of the cell such that the conformation of IMPs is highly dynamic. Hence monitoring real time conformational changes in these proteins by NMR and x-ray methods is unfeasible, and alternative methods are needed.Recent progress in MS has enabled a novel MS-based protein oxidative footprinting technique to determine structural information by mapping of oxidation induced by hydroxyl (OH) radicals. This method is an adaptation of the OH radical footprinting first developed by Tullius and co-workers (2, 3) for the folding study of DNA/RNA molecules in solution. Several groups have since extended this method in combination with mass spectrometry for the mapping of a protein surface (4–8). In these studies, OH radicals oxidize amino acid residues located on the protein surface and produce stable covalent modifications to side chains without causing backbone cleavages. Due to the very small size and nonspecific activity of hydroxyl radicals, this is a random process dependent only on the solvent-accessible surface and the chemical properties of the exposed amino acids. It has been reported that there are about 12 possible types of side-chain oxidation products in protein footprinting experiments (9); however, not all of these oxidation products are common events as reviewed. The most common event results in formation of an alcohol group for almost all the amino acid residues with mass increases of +16 Da. Another common event is the formation of an aldehyde/ketone group for eight residues, Val, Ile, Leu, Lys, Arg, Pro, Glu, and Gln, with mass increases of +14 Da (9). Others include +32 Da on Cys, Met, Trp, Phe, and Tyr; +48 Da on Cys and Trp; +5, −10, −22, and −23 Da on His; −30 Da on Asp and Glu; −16 Da on Cys; −32 Da on Met; and −2 Da on Ser and Thr (9).The oxidized protein is subsequently sequenced by MS/MS to locate the oxidized amino acid residues. The oxidized amino acid residues provide the surface information of the protein, and thus the surface topology can be mapped. At the same time, the oxidation level of each side chain can be accurately measured by quantitative liquid chromatography-coupled MS. Because the level of oxidation for each tryptic peptide depends primarily on the solvent accessibility of the peptide side chains that is in turn dependent on the conformation of the protein, the oxidation level and changes in its level for each peptide can therefore be used to determine conformation and conformational changes of the protein. This approach has been shown to be a powerful technique in understanding ligand-induced conformational changes when coupled with existing structural data that are either experimentally or computationally generated (10).Despite recent advances, most current surface mapping techniques still require purified proteins and are therefore not amenable to the conformation determination of most IMPs or their complexes. In particular, the current techniques still cannot determine real time in vivo structural changes of IMPs that are important in understanding the functions of IMPs. Therefore, the structural dynamics of IMP remain elusive.IMPs display two membrane-spanning tertiary structural motifs, i.e. α-helix bundles in the cytoplasmic membrane and β-barrels in the outer membrane (11). In Gram-negative bacteria, the outer membrane forms a protective permeability barrier around the cells and serves as a molecular filter for hydrophilic substances (12). To facilitate this, channel-forming proteins are embedded in the outer membrane to mediate the transport of nutrients and ions across the membrane into the periplasm. The most abundant proteins in this class are the porins that form aqueous passive channels with a physical diameter of about 1 nm for the transport of ions and relatively small polar molecules across the outer membrane of bacteria (13–15). Porins form β-barrels with 16 or 18 membrane-spanning antiparallel β-strands that are connected by short turns on the periplasmic side named T1, T2, etc. and long loops on the extracellular side named L1, L2, etc. (16, 17). In all porins, the constriction at the barrel center is formed by an inserted loop L3 (13, 16, 18), which is not exposed to the cell surface but folds back into the channel and contributes significantly to the permeability of the pore. Functional porins are homotrimers of these β-barrel subunits with each subunit producing a channel in the outer membrane, and the trimer therefore contains three channels (14). Currently known porins fall into two distinct groups: 16-stranded general diffusion porins such as the matrix porin OmpF that transports ions and small molecules (<600 Da) without much selectivity and 18-stranded specific porins such as maltoporin that have a precise selectivity for a defined substrate (15, 17, 19–22). General porin trimers exhibit symmetrical or asymmetrical voltage gating when reconstituted into planar lipid bilayers in electrophysiological studies (23–27). By measuring the ion conductance in vitro, porin trimers were found to be able to exhibit at least two functional states, an open and a closed state, in response to changes in the transmembrane potential difference, known as “voltage gating”, and the voltage above which the porin channel will be closed is called the critical voltage, Vc. Although existing data generally support voltage gating in vitro, there are no data to support voltage gating in vivo (25, 27).We describe here a novel adaptation of an oxidative footprinting and MS technique to study in vivo conformational changes of IMPs in living cells. We used Escherichia coli as a model to study the structural dynamics of the outer membrane proteins with an emphasis on the matrix porin OmpF. Considering that the yellowish and cloudy E. coli bacteria culture would interfere with the laser photolysis of H₂O₂ because H₂O₂ only absorbs laser light at a wavelength around 250 nm and this cloudy medium would inhibit the laser penetration (4), we adopted a Fenton reaction but not the pulsed laser photolysis method to oxidize the living bacteria. Fenton oxidation is not guaranteed for many proteins, especially the highly dynamic proteins, because this method needs a relatively longer incubation time (2, 4, 5, 9). This time scale is too long because most of the conformational changes of macromolecules take place only in the time scale of milliseconds to seconds (10). However, Fenton chemistry is suitable to study the outer membrane porins because they have extremely stable structures that are restricted by both the intramolecular hydrogen bond and the hydrophobic interaction with the lipid bilayer wall, and a large conformational change of this kind of protein is considered unlikely (14, 20).In this method, living E. coli cells were first exposed to OH radicals generated from an in situ Fenton reaction between hydrogen peroxide and Fe(II)-bound EDTA. The outer membrane proteins that have a large solvent-accessible surface area would be preferentially oxidized by the OH radicals. The outer membrane proteins were then isolated and enriched from the cell lysate. The porin OmpF was subsequently purified by 1D SDS-PAGE electrophoresis followed by proteolysis to generate peptides for analysis and identification by LC-MS/MS and bioinformatics analysis. The structural information of porin OmpF as interpreted from our oxidative footprinting and MS study was in agreement with the x-ray crystallography structure (20). Highly reproducible oxidized amino acid residues were widely distributed in the external loops, transmembrane β-strands, and periplasmic turnings of the protein, and all of them were validated as solvent-accessible according to the x-ray structure of porin OmpF.We then extended this method to probe for voltage gating of porin OmpF under different conditioned circumstances to assess the robustness of its application in complicated biological systems. Here E. coli was stimulated first with either a low ionic strength solution or a low pH buffer after harvest and exposed to OH radicals generated by Fenton reagents in solution for 3 min. Our data showed that the extent of oxidation of two polypeptides from extracellular loops remained constant independent of conditions that induced either channel closing or opening. However, we observed that oxidation level of peptides in β-stranded areas deep inside the porin channel was reduced when the environment changed from one that induced channel opening to another that induced channel closing. These data not only supported the voltage gating of porin OmpF in vivo but also revealed the molecular basis underlying voltage gating of porin OmpF.     

Decomposition Analysis of Competitive Symmetry and Size Structure Dynamics

An analysis is introduced, based on the decomposition of relativegrowth rates, to examine the mode of competition (i.e. whethercompetition is symmetric or asymmetric), the size-dependenceof growth, and their interdependence. In particular, the basisfor two commonly held views is examined: (1) that the type ofresource limitation determines the mode of competition, and(2) that asymmetric competition always leads to size-divergencebetween unequal competitors. It is shown that in field-grownmillet plants, competition for light was symmetric at low densityand asymmetric at high density. However, size variation at lowdensity decreased during growth, because small plants had greaterrelative growth rates than larger plants. Size variation stayedconstant at high density, since plants of all sizes had equalaverage relative growth rates. Based on these results and ageneral discussion, it is proposed that the type of resourcelimitation does not determine the mode of competition. Competitionfor light can be symmetric, and foraging for heterogeneouslydistributed soil resources can produce asymmetric competitionbelow-ground. Furthermore, the mode of competition alone doesnot determine size structure dynamics. Size-dependence of resourceconversion efficiency and allocation can modify the effectsof resource uptake on growth. Pennisetum americanum‘Custer ’; mode of competition; size structure dynamics; plant growth analysis     

Regulation of Structural Dynamics within a Signal Recognition Particle Promotes Binding of Protein Targeting Substrates

Protein targeting is critical in all living organisms and involves a signal recognition particle (SRP), an SRP receptor, and a translocase. In co-translational targeting, interactions among these proteins are mediated by the ribosome. In chloroplasts, the light-harvesting chlorophyll-binding protein (LHCP) in the thylakoid membrane is targeted post-translationally without a ribosome. A multidomain chloroplast-specific subunit of the SRP, cpSRP43, is proposed to take on the role of coordinating the sequence of targeting events. Here, we demonstrate that cpSRP43 exhibits significant interdomain dynamics that are reduced upon binding its SRP binding partner, cpSRP54. We showed that the affinity of cpSRP43 for the binding motif of LHCP (L18) increases when cpSRP43 is complexed to the binding motif of cpSRP54 (cpSRP54_pep). These results support the conclusion that substrate binding to the chloroplast SRP is modulated by protein structural dynamics in which a major role of cpSRP54 is to improve substrate binding efficiency to the cpSRP.     

细胞结构动力学——实时调控细胞力学的因素分析

机械力普遍存在于活细胞的生命活动中,而细胞内力学活动必须依赖骨架结构传递,这种独特的力学形式被称为细胞结构力学.单位时间内细胞结构力学变化受多因素调控,如外力、渗透压、动力分子、张力敏感性离子通道、胞内力学感受器及骨架组装等,构成了细胞结构动力学研究的重要内容.基于荧光共振能量转移(FRET)原理开发的荧光张力探针能整合到细胞骨架内,将细胞结构力学变化转化为光学信号,可能带来细胞力学研究的革命.随着细胞结构动力学研究内容的不断深入,特别是太空时代细胞力学稳态的打破,细胞结构动力学将在生命及医学研究领域显露出越来越重要的地位.     

Methionine Mutations of Outer Membrane Protein X Influence Structural Stability and Beta-Barrel Unfolding

We report the biochemical and biophysical characterization of outer membrane protein X (OmpX), an eight-stranded transmembrane β-barrel from E. coli, and compare the barrel behavior with a mutant devoid of methionine residues. Transmembrane outer membrane proteins of bacterial origin are known to display high tolerance to sequence rearrangements and mutations. Our studies with the triple mutant of OmpX that is devoid of all internal methionine residues (M18L; M21L; M118L) indicate that Met replacement has no influence on the refolding efficiency and structural characteristics of the protein. Surprisingly, the conserved substitution of Met→Leu leads to barrel destabilization and causes a lowering of the unfolding free energy by a factor of ∼8.5 kJ/mol, despite the mutations occurring at the loop regions. We report that the barrel destabilization is accompanied by a loss in cooperativity of unfolding in the presence of chemical denaturants. Furthermore, we are able to detect an unfolding intermediate in the Met-less barrel, whereas the parent protein exhibits a classic two-state unfolding. Thermal denaturation measurements also suggest a greater susceptibility of the OmpX barrel to heat, in the Met-less construct. Our studies reveal that even subtle variations in the extra-membrane region of rigid barrel structures such as OmpX, may bear severe implications on barrel stability. We propose that methionines contribute to efficient barrel structuring and protein-lipid interactions, and are therefore important elements of OmpX stability.     

Anti-CRISPR蛋白AcrVA2的结构生物学研究

大多数古生菌及半数细菌都含有成簇有规律间隔的短回文重复序列(clustered regularly interspaced short palindromic repeats,CRISPR)和CRISPR相关(CRISPR-associated,Cas)蛋白质构成的适应性免疫系统,来抵御外界噬菌体的入侵.而噬菌体为了对抗这种免疫系统,也进化出许多抗CRISPR (anti-CRISPR,Acr)的蛋白质,使得CRISPR-Cas系统受到抑制.来自牛眼莫拉氏菌(Moraxella bovoculi)的AcrVA2是目前发现的可抑制V-A型CRISPR-Cas系统效应蛋白Cas12a发挥切割活性的Acr蛋白之一,其作用机理尚不清楚.本文解析了自由状态的AcrVA2和MbCas12a^620-636-AcrVA2复合物的晶体结构,发现AcrVA2蛋白采用了一种新的α-β折叠结构,且只与自由状态的Cas12a结合.此外,AcrVA2与MbCas12a^620-636的结合主要依靠氢键和盐桥的相互作用力,并通过疏水界面得到进一步稳定.这些结果提示,AcrV...     

蛋白质磷酸化作用的结构基础

蛋白质可逆磷酸化涉及到几乎所有细胞活动的调节.着重探讨了影响蛋白激酶作用专一性的几个因素和磷酸化影响蛋白质功能的结构基础及作用机制.     

基于SWISS-MODEL的蛋白质三维结构建模

蛋白质的三级结构预测可通过同源建模、Threading和TOPITS等方法进行,但同源建模是应用最为广泛的方法。SWISS-MODEL正是一个基于同源建模的蛋白质结构服务器。它与ExPASy网站和DeepView程序是紧密相联系的。该文重点介绍SWISS-MODEL的提交方式、建模的步骤、结果的评估和应用程序等。     

放牧对草场植被动态的影响

放牧对草场植被动态的影响吴德东,周景荣,朱德华,刘淑玲（辽宁省固沙造林研究所,章古台１２３２０３）王志海,马占信,赵建平（阜新市阿尔乡畜牧场,１２３２０４）ＩｎｆｌｕｅｎｃｅｏｆＧｒａｚｉｎｇｏｎＶｅｇｅｔａｔｉｏｎＤｙｎａｍｉｃｓｏｎＰａｓｔｕｒｅ...