An incipient 3(10) helix in Piv-Pro-Pro-Ala-NHMe as a model for peptide folding

The molecular mechanism of helix nucleation in peptides and proteins is not yet understood and the question of whether sharp turns in the polypeptide backbone serve as nuclei for protein folding has evoked controversy. A recent study of the conformation of a tetrapeptide containing the stereochemically constrained residue alpha-aminoisobutyric acid, both in solution and the solid state, yielded a structure consisting of two consecutive beta-turns, leading to an incipient 3(10) helical conformation. :This led us to speculate that specific tri- and tetrapeptide sequences may indeed provide a helical twist to the amino-terminal segment of helical regions in proteins and provide a nucleation site for further propagation. The transformation from a 3(10) helical structure to an alpha-helix should be facile and requires only small changes in the phi and psi conformational angles and a rearrangement of the hydrogen bonding pattern. If such a mechanism is involved then it should be possible to isolate an incipient 3(10) helical conformation in a tripeptide amide or tetrapeptide sequence, based purely on the driving force derived from short-range interactions. We have synthesised and studied the model peptide pivaloyl-Pro-Pro-Ala-NHMe (compound I) and provide here spectroscopic evidence for a 3(10) helical conformation in compound I.     

Respective roles of short-and long-range interactions in protein folding

A new method was presented to discuss the respective roles of short- and long-range interactions in protein folding. It‘s based on an off-lattice model, which is also being called as toy model. Simulated annealing algorithm was used to search its native conformation. When it is applied to analysis proteins lagt and laho, we find that helical segment catmot fold into native conformation without the influence of long-range interactions. That‘s to say that long-range interactions are the main determinants in protein folding.     

丙氨酸甘氨酸混合三、四肽构象稳定性的快速预测

使用特殊氢方法预测丙氨酸甘氨酸混合三肽和四肽分子共111个构象的相对稳定性,并与B3LYP/6-31G^＊方法的相对能量比较,得到了满意的结果.在所有111个构象中,特殊氢方法相对能量与B3LYP/6-31G^＊方法相对能量偏差绝对值大于2.0 kcal/mol的只有3个,偏差在1.0～2.0 kcal/mol之间的有18个,其余均小于1.0 kcal/mol.本文结果表明特殊氢原子在决定多肽构象稳定性方面的内秉重要性.     

Vinculin activation by talin through helical bundle conversion

Vinculin is a conserved component and an essential regulator of both cell-cell (cadherin-mediated) and cell-matrix (integrin-talin-mediated focal adhesions) junctions, and it anchors these adhesion complexes to the actin cytoskeleton by binding to talin in integrin complexes or to alpha-actinin in cadherin junctions. In its resting state, vinculin is held in a closed conformation through interactions between its head (Vh) and tail (Vt) domains. The binding of vinculin to focal adhesions requires its association with talin. Here we report the crystal structures of human vinculin in its inactive and talin-activated states. Talin binding induces marked conformational changes in Vh, creating a novel helical bundle structure, and this alteration actively displaces Vt from Vh. These results, as well as the ability of alpha-actinin to also bind to Vh and displace Vt from pre-existing Vh-Vt complexes, support a model whereby Vh functions as a domain that undergoes marked structural changes that allow vinculin to direct cytoskeletal assembly in focal adhesions and adherens junctions. Notably, talin's effects on Vh structure establish helical bundle conversion as a signalling mechanism by which proteins direct cellular responses.     

Structural architecture of an outer membrane channel as determined by electron crystallography.

Porins are a family of membrane channels commonly found in the outer membranes of Gram-negative bacteria where they serve as diffusional pathways for waste products, nutrients and antibiotics, and can also be receptors for bacteriophages. Porin channels have been shown in vitro to be voltage-gated. They can exhibit slight selectivities for certain solutes; for example PhoE porin has some selectivity for anionic and phosphate-containing compounds. Unlike many known membrane proteins which often contain long stretches of hydrophobic segments that are believed to traverse the membrane in a helical conformation, porins are found to have charged residues distributed almost uniformly along their primary sequences and have most of their secondary structure in a beta-sheet conformation. We have made crystalline patches of PhoE porin embedded in a lipid bilayer and have used these to determine the structure of PhoE porin by electron crystallography to a resolution of 6A. The basic structure consists of a trimer of elliptically shaped, cylindrical walls of beta sheet. Each cylinder has an inner lining, formed by parts of the polypeptide, that defines the channel size. The structure provides a clue as to how deletions of segments of polypeptide, which are found in certain mutants, can result in an actual increase in the channel size.     

Complete atomic model of the bacterial flagellar filament by electron cryomicroscopy

The bacterial flagellar filament is a helical propeller for bacterial locomotion. It is a helical assembly of a single protein, flagellin, and its tubular structure is formed by 11 protofilaments in two distinct conformations, L- and R-type, for supercoiling. The X-ray crystal structure of a flagellin fragment lacking about 100 terminal residues revealed the protofilament structure, but the full filament structure is still essential for understanding the mechanism of supercoiling and polymerization. Here we report a complete atomic model of the R-type filament by electron cryomicroscopy. A density map obtained from image data up to 4 A resolution shows the feature of alpha-helical backbone and some large side chains. The atomic model built on the map reveals intricate molecular packing and an alpha-helical coiled coil formed by the terminal chains in the inner core of the filament, with its intersubunit hydrophobic interactions having an important role in stabilizing the filament.     

聚皂溶液及其紫外-可见探针光谱研究

缩聚法合成了四乙烯五胺－环氧氯丙烷型聚皂（ＨＰＴＥ）．粘度法的研究表明,ＨＰＴＥ在水溶液中的构象变化与其分子链上疏水烷基形成的疏水微区有关．以甲基橙为显色探针的紫外－可见光谱研究表明,ＨＰＴＥ与甲基橙的相互作用是分子间（内）的静电吸引和疏水相互作用的共同结果,甲基橙增溶于ＨＰＴＥ的疏水微区中,改变了甲基橙分子的微环境极性,引起甲基橙分子λｍａｘ的蓝移．     

几种盐类对光系统Ⅱ33kD锰稳定蛋白溶液构象的影响

借助内源荧光和圆二色谱分析，考察了几种不同的盐溶液（CaCl2,NaCl,脲，三氯乙酸、二硫苏糖醇）对水溶液中33kD蛋白构象的影响，以295nm波长的乐激发时，33kD蛋白具有330nm的荧光发射峰，表明33kD蛋白所含唯一的^241Trp位于分子内部的疏水部位，CaCl2、脲、三氯乙酸或二硫苏糖醇的存在，均会改变33kD蛋白的内源荧光的强度和位置，而NaCl几科无影响，脲、二硫苏糖醇对33kD蛋白的二级结构的影响较大，因此维系33kD蛋白构象的主要因素包括：二硫键、疏水作用、范得华力和氢键，而离子键的贡献较小。     

Two-headed binding of a processive myosin to F-actin

Myosins are motor proteins in cells. They move along actin by changing shape after making stereospecific interactions with the actin subunits. As these are arranged helically, a succession of steps will follow a helical path. However, if the myosin heads are long enough to span the actin helical repeat (approximately 36 nm), linear motion is possible. Muscle myosin (myosin II) heads are about 16 nm long, which is insufficient to span the repeat. Myosin V, however, has heads of about 31 nm that could span 36 nm and thus allow single two-headed molecules to transport cargo by walking straight. Here we use electron microscopy to show that while working, myosin V spans the helical repeat. The heads are mostly 13 actin subunits apart, with values of 11 or 15 also found. Typically the structure is polar and one head is curved, the other straighter. Single particle processing reveals the polarity of the underlying actin filament, showing that the curved head is the leading one. The shape of the leading head may correspond to the beginning of the working stroke of the motor. We also observe molecules attached by one head in this conformation.     

Structural changes in glycogen phosphorylase induced by phosphorylation

A comparison of the refined crystal structures of dimeric glycogen phosphorylase b and a reveals structural changes that represent the first step in the activation of the enzyme. On phosphorylation of serine-14, the N-terminus of each subunit assumes an ordered helical conformation and binds to the surface of the dimer. The consequent structural changes at the N- and C-terminal regions lead to strengthened interactions between subunits and alter the binding sites for allosteric effectors and substrates.     

The effect of α-helix comformation on interaction between model oligopeptides and polymers

The study of interactions between peptides and synthetic polymers has aroused considerable interest recently. In the present paper, the interaction mechanism between adsorbent DMAPAM and two peptides, VVRGCTWW（V8） and CTWW（C4）, was investigated at molecular level by NMR and molecular stimulation. The results demonstrated that the conformation of oligopeptides played a vital role in their adsorption capacity on the adsorbent. Weak interactions could incorporate with each other between V8 and DMAPAM because there was a hydrophobic cleavage in o-helix conformation of V8. So the ad- sorption capacity of DMAPAM to V8 was higher. This was significant to explore the interaction mechanism between peptides and biomaterials.     

Solution structure of the major binding protein for the immunosuppressant FK506

The major FK506 binding protein (FKBP, relative molecular mass approximately 11,800; Mr 11.8K) and cyclophilin (Mr approximately 17K) belong to a class of proteins termed immunophilins. Although unrelated at the amino-acid sequence level, they both possess peptidyl-prolyl cis-trans isomerase activities which are inhibited by immunosuppressants that block signal transduction pathways leading to T-lymphocyte activation. FK506 and rapamycin strongly inhibit the peptidyl-prolyl cis-trans isomerase activity of FKBP, whereas cyclosporin A inhibits that of cyclophilin. The significance of this enzyme activity and the role of the immunophilins in immunoregulation is unknown. To understand better the function of the immunophilins and their interaction with inhibitors, we are investigating the solution structures of FKBP and FKBP-inhibitor complexes by multidimensional NMR methods. Here we report the solution conformation of FKBP, as generated by NMR, distance geometry and molecular dynamics methods. The regular secondary structure of FKBP is composed mainly of beta sheet (approximately 35%) with little helical structure (less than 10%). The hydrophobic core of the molecule, containing the buried side chains of six of the protein's nine aromatic amino acids, is enclosed by a five-stranded antiparallel beta sheet on one side, a loop and a short helix at residues 51-56 and 57-65, and an aperiodic loop at residues 81-95. Examination of the structure suggests a possible site of interaction with FK506.     

Orthorhombic crystal structure of an insulin mutant B9Asp at 0.20 nm resolution

Human B9(Ser~i'Asp) insulin, a fast-absorption insulin analogue, was obtained by site-directed mutagenesis at B9 position. The orthorhombic crystal structure of B9Asp insulin was analyzed by crystallography at 0.20 nm resolution. Although no significant change of its overall conformation was observed, the local conformation flanking B9 site differed greatly from native T6 human insulin. The substitution of serine at B9 position by aspartic acid resulted in obvious alteration of local hydrophobic and hydrophilic interactions. As a result, the insulin dimer became unstable and the capability of the hexamer formation was diminished extensively. All these properties contribute to the fast-absorption of B9Asp, In addition, the open state of N-terminus of B-chain, which differs from T- or R- state, might suggest a new conformational state in the monomer or dimer insulin.     

Insulin trigger, cyclic AMP-dependent activation and phosphorylation of a plasma membrane cyclic AMP phosphodiesterase

Regulation of blood glucose levels by the liver is primarily achieved by the action of two peptide hormones, insulin and glucagon, which bind to specific receptors associated with the hepatocyte plasma membrane. Whilst the molecular action of glucagon at the level of the cell plasma membrane in activating adenylate cyclase is relatively well understood, we know little, if anything, of the molecular consequences of insulin occupying its receptor. We demonstrate here that insulin, at physiologically relevant concentrations, can trigger the cyclic AMP-dependent activation and phosphorylation of a low Km cyclic AMP phosphodiesterase attached to the liver plasma membrane. Such an effect may in part explain the ability of insulin to inhibit the increase in cellular cyclic AMP content that glucagon alone produces by activation of adenylate cyclase. Our observation that basal, intracellular cyclic AMP levels are insufficient to allow insulin to activate the cyclic AMP phosphodiesterase, yet those cyclic AMP levels achieved after exposure of the cells to glucagon are sufficient, gives a molecular rationale to Butcher and Sutherland's proposal that it is necessary to first elevate cellular cyclic AMP levels before they can be depressed by insulin.     

分子模拟研究β-环糊精整体柱的分离识别机理

利用分子动力学模拟和色谱分析的方法研究了葛根素、大豆甙和大豆苷元在β-环糊精配基键合聚甲基丙烯酸缩水甘油酯整体柱上的分离识别机理。分子模拟结果显示,β-环糊精与客体分子的结合能越大,β-环糊精与客体小分子的结合作用越大,形成的包合物相对越稳定,则在色谱分离中客体分子在固定相上的保留因子越大,在洗脱顺序中表现为后洗脱出来。通过模拟计算β-环糊精与客体分子的结合能和氢键作用的大小,可以成功地预测客体小分子在β-环糊精配基整体柱上的保留行为和洗脱顺序。利用质量作用模型,通过改变色谱流动相中添加剂的浓度,证明了葛根素、大豆甙和大豆苷元在β-环糊精配基整体柱上的保留符合氢键和疏水作用共存的混合作用模式。     

Molecular structure of r(GCG)d(TATACGC): a DNA--RNA hybrid helix joined to double helical DNA

The molecule r(GCG)d(TATACGC) is self-complementary and forms two DNA--RNA hybrid segments surrounding a central region of double helical DNA; its molecular structure has been solved by X-ray analysis. All three parts of the molecule adopt a conformation which is close to that seen in the 11-fold RNA double helix. The conformation of the ribonucleotides is partly determined by water molecules bridging between the ribose O2' hydroxyl group and cytosine O2. The hybrid-DNA duplex junction contains no structural discontinuities. However, the central DNA TATA sequence has some structural irregularities.     

嵌段共聚水溶性高分子在空气/水界面吸附态的构象

对于用几种不同疏水／亲水单体比合成的聚(丙烯酸丁酯／丙烯酸)共聚物样品测定了其水溶液的表面张力,运用Gibbs吸附方程求得在样品溶液临界胶束浓度时样品在气／液界面的分子吸附面积,以此推测其吸附态构象。结果表明,亲水单体单元在共聚物中含量多时,样品分子趋于在水中溶解;疏水单体单元多时,样品分子趋于在表面吸附。其吸附态构象为分子链上相邻的数个丁酯侧基吸附于水面,形成“薄煎饼状”疏水微区。     

Structure of the parainfluenza virus 5 F protein in its metastable, prefusion conformation

Enveloped viruses have evolved complex glycoprotein machinery that drives the fusion of viral and cellular membranes, permitting entry of the viral genome into the cell. For the paramyxoviruses, the fusion (F) protein catalyses this membrane merger and entry step, and it has been postulated that the F protein undergoes complex refolding during this process. Here we report the crystal structure of the parainfluenza virus 5 F protein in its prefusion conformation, stabilized by the addition of a carboxy-terminal trimerization domain. The structure of the F protein shows that there are profound conformational differences between the pre- and postfusion states, involving transformations in secondary and tertiary structure. The positions and structural transitions of key parts of the fusion machinery, including the hydrophobic fusion peptide and two helical heptad repeat regions, clarify the mechanism of membrane fusion mediated by the F protein.     

Structural basis for the activation of anthrax adenylyl cyclase exotoxin by calmodulin.

Oedema factor, a calmodulin-activated adenylyl cyclase, is important in the pathogenesis of anthrax. Here we report the X-ray structures of oedema factor with and without bound calmodulin. Oedema factor shares no significant structural homology with mammalian adenylyl cyclases or other proteins. In the active site, 3'-deoxy-ATP and a single metal ion are well positioned for catalysis with histidine 351 as the catalytic base. This mechanism differs from the mechanism of two-metal-ion catalysis proposed for mammalian adenylyl cyclases. Four discrete regions of oedema factor form a surface that recognizes an extended conformation of calmodulin, which is very different from the collapsed conformation observed in other structures of calmodulin bound to effector peptides. On calmodulin binding, an oedema factor helical domain of relative molecular mass 15,000 undergoes a 15 A translation and a 30 degrees rotation away from the oedema factor catalytic core, which stabilizes a disordered loop and leads to enzyme activation. These allosteric changes provide the first molecular details of how calmodulin modulates one of its targets.