Syncephalastrum contaminatum,a new species in the Mucorales from Australia

A new species is described in the Mucorales family Syncephalastraceae: Syncephalastrum contaminatum, isolated as an in vitro culture from a laboratory contaminant. The species has variable copies of the internal transcribed spacer (ITS) regions, requiring cloning of these regions prior to Sanger sequencing before subsequent use in phylogenetic comparisons with other fungi. The genome of the strain was sequenced using short paired-reads to yield a draft genome of 28.6 Mb. Syncephalastrum contaminatum is distinguished by diverse DNA sequences at several loci from the other species of Syncephalastrum, including only 81% sequence identity with its ITS regions to that of S. racemosum. Its merosporangium produces four or more asexual spores and the genome sequencing information suggests that the species is heterothallic. The identification of this species highlights the limited knowledge about the early lineages of fungi both in Australia and globally.     

A Bayes-optimal sequence-structure theory that unifies protein sequence-structure recognition and alignment

A rigorous Bayesian analysis is presented that unifies protein sequence-structure alignment and recognition. Given a sequence, explicit formulae are derived to select (1) its globally most probable core structure from a structure library; (2) its globally most probable alignment to a given core structure; (3) its most probable joint core structure and alignment chosen globally across the entire library; and (4) its most probable individual segments, secondary structure, and super-secondary structures across the entire library. The computations involved are NP-hard in the general case (3D-3D). Fast exact recursions for the restricted sequence singleton-only (1D-3D) case are given. Conclusions include: (a) the most probable joint core structure and alignment is not necessarily the most probable alignment of the most probable core structure, but rather maximizes the product of core and alignment probabilities; (b) use of a sequence-independent linear or affine gap penalty may result in the highest-probability threading not having the lowest score; (c) selecting the most probable core structure from the library (core structure selection or fold recognition only) involves comparing probabilities summed over all possible alignments of the sequence to the core, and not comparing individual optimal (or near-optimal) sequence-structure alignments; and (d) assuming uninformative priors, core structure selection is equivalent to comparing the ratio of two global means.     

Protein loop structure prediction with flexible stem geometries

The structure prediction of loops with flexible stem residues is addressed in this article. While the secondary structure of the stem residues is assumed to be known, the geometry of the protein into which the loop must fit is considered to be unknown in our methodology. As a consequence, the compatibility of the loop with the remainder of the protein is not used as a criterion to reject loop decoys. The loop structure prediction with flexible stems is more difficult than fitting loops into a known protein structure in that a larger conformational space has to be covered. The main focus of the study is to assess the precision of loop structure prediction if no information on the protein geometry is available. The proposed approach is based on (1) dihedral angle sampling, (2) structure optimization by energy minimization with a physically based energy function, (3) clustering, and (4) a comparison of strategies for the selection of loops identified in (3). Steps (1) and (2) have similarities to previous approaches to loop structure prediction with fixed stems. Step (3) is based on a new iterative approach to clustering that is tailored for the loop structure prediction problem with flexible stems. In this new approach, clustering is not only used to identify conformers that are likely to be close to the native structure, but clustering is also employed to identify far-from-native decoys. By discarding these decoys iteratively, the overall quality of the ensemble and the loop structure prediction is improved. Step (4) provides a comparative study of criteria for loop selection based on energy, colony energy, cluster density, and a hybrid criterion introduced here. The proposed method is tested on a large set of 3215 loops from proteins in the Pdb-Select25 set and to 179 loops from proteins from the Casp6 experiment.     

Structure and mechanism of proton-translocating transhydrogenase

Recent developments have led to advances in our understanding of the structure and mechanism of action of proton-translocating (or AB) transhydrogenase. There is (a) a high-resolution crystal structure, and an NMR structure, of the NADP(H)-binding component (dIII), (b) a homology-based model of the NAD(H)-binding component (dI) and (c) an emerging consensus on the position of the transmembrane helices (in dII). The crystal structure of dIII, in particular, provides new insights into the mechanism by which the energy released in proton translocation across the membrane is coupled to changes in the binding affinities of NADP(+) and NADPH that drive the chemical reaction.     

The role of irregular unit,GAAS, on the secondary structure of Bombyx mori silk fibroin studied with 13C CP/MAS NMR and wide-angle X-ray scattering

Bombyx mori silk fibroin is a fibrous protein whose fiber is extremely strong and tough, although it is produced by the silkworm at room temperature and from an aqueous solution. The primary structure is mainly Ala-Gly alternative copolypeptide, but Gly-Ala-Ala-Ser units appear frequently and periodically. Thus, this study aims at elucidating the role of such Gly-Ala-Ala-Ser units on the secondary structure. The sequential model peptides containing Gly-Ala-Ala-Ser units selected from the primary structure of B. mori silk fibroin were synthesized, and their secondary structure was studied with (13)C CP/MAS NMR and wide-angle X-ray scattering. The (13)C isotope labeling of the peptides and the (13)C conformation-dependent chemical shifts were used for the purpose. The Ala-Ala units take antiparallel beta-sheet structure locally, and the introduction of one Ala-Ala unit in (Ala-Gly)(15) chain promotes dramatical structural changes from silk I (repeated beta-turn type II structure) to silk II (antiparallel beta-sheet structure). Thus, the presence of Ala-Ala units in B. mori silk fibroin chain will be one of the inducing factors of the structural transition for silk fiber formation. The role of Tyr residue in the peptide chain was also studied and clarified to induce "locally nonordered structure."     

厌氧氨氧化菌火山口结构

厌氧氨氧化(anaerobic ammonium oxidation, anammox)是微生物学、地质学和环境学领域的重要反应,厌氧氨氧化菌(anaerobic ammonium-oxidizing bacteria, AnAOB)是厌氧氨氧化的驱动器,探明AnAOB的生物学性状对厌氧氨氧化的应用具有重要意义。火山口结构是AnAOB的标志性微观结构,也是AnAOB的重要识别特征。由于迄今没有获得AnAOB纯培养物,相关研究进展缓慢。本文对AnAOB及其所归属的浮霉状菌的火山口结构研究进展作了综述,探讨了火山口结构的形态特征、生理功能和生态意义,得出以下结论:(1) AnAOB的火山口结构均匀分布在细胞表面,其直径约5 nm;(2) AnAOB的火山口结构推测向外可连通细胞外膜和内膜,向内可与厌氧氨氧化体膜相连,对于物质转运及转化具有重要意义;(3)火山口结构具有遗传稳定性,其形成可能与鞭毛脱落相关;(4) AnAOB的火山口结构可能通过促进细胞物质交流、信息通讯等在维持其生态位稳定方面起作用。     

Jararhagin-derived RKKH peptides induce structural changes in alpha1I domain of human integrin alpha1beta1

Integrin alpha(1)beta(1) is one of four collagen-binding integrins in humans. Collagens bind to the alphaI domain and in the case of alpha(2)I collagen binding is competitively inhibited by peptides containing the RKKH sequence and derived from the metalloproteinase jararhagin of snake venom from Bothrops jararaca. In alpha(2)I, these peptides bind near the metal ion-dependent adhesion site (MIDAS), where a collagen (I)-like peptide is known to bind; magnesium is required for binding. Published structures of the ligand-bound "open" conformation of alpha(2)I differs significantly from the "closed" conformation seen in the structure of apo-alpha(2)I near MIDAS. Here we show that two peptides, CTRKKHDC and CARKKHDC, derived from jararhagin also bind to alpha(1)I and competitively inhibit collagen I binding. Furthermore, calorimetric and fluorimetric measurements show that the structure of the complex of alpha(1)I with Mg(2+) and CTRKKHDC differs from structure in the absence of peptide. A comparison of the x-ray structure of apo-alpha(1)I ("closed" conformation) and a model structure of the alpha(1)I ("open" conformation) based on the closely related structure of alpha(2)I reveals that the binding site is partially blocked to ligands by Glu(255) and Tyr(285) in the "closed" structure, whereas in the "open" structure helix C is unwound and these residues are shifted, and the "RKKH" peptides fit well when docked. The "open" conformation of alpha(2)I resulting from binding a collagen (I)-like peptide leads to exposure of hydrophobic surface, also seen in the model of alpha(1)I and shown experimentally for alpha(1)I using a fluorescent hydrophobic probe.     

Molecular dynamics simulations of the native and partially folded states of ubiquitin: influence of methanol cosolvent, pH, and temperature on the protein structure and dynamics

A series of explicit-solvent molecular dynamics simulations of the protein ubiquitin are reported, which investigate the effect of environmental factors (presence of methanol cosolvent in the aqueous solution, neutral or low pH value, room or elevated temperature) on the structure, stability, and dynamics of the protein. The simulations are initiated either from the native structure of the protein or from a model of a partially folded state (A-state) that is known to exist at low pH in methanol-water mixtures. The main results of the simulations are: (1) The ubiquitin native structure is remarkably stable at neutral pH in water; (2) the addition of the methanol cosolvent enhances the stability of the secondary structure but weakens tertiary interactions within the protein; (3) this influence of methanol on the protein structure is enhanced at low pH, while the effect of lowering the pH in pure water is limited; and (4) the A-state of ubiquitin can be described as a set of relatively rigid secondary structure elements (a native-like beta-sheet and native-like alpha-helix plus two nonnative alpha-helices) connected by flexible linkers.     

Packing of transmembrane helices in bacteriorhodopsin folding: structure and thermodynamics

We propose a coarse-grained (CG) model to study the native structure and physical properties of helical membrane proteins (HMPs) using off-lattice computer simulations. Instead of considering sequence heterogeneity explicitly, we model its effect on the packing of helices by employing a mean packing parameter r(0), which is calculated from an all-atom (AA) model. Specifically, this CG model is applied to investigate the packing of helices in bacteriorhodopsin (BR), and predicts the seven helix bundle structure of BR with a root mean square deviation (RMSD) in coordinates of helix backbone atoms (N, C, C(alpha)) of 3.99 A from its crystal structure. This predicted structure is further refined in an AA model by Amber and the refined structure has a RMSD (in coordinates of helix backbone atoms) of 2.64 A. The predicted packing position, tilting angle, and orientation angle of each helix in the refined structure are consistent with experimental data and their physical origins can be well understood in our model. Our results show that a reasonably good structure of BR can be predicted by using such a dual-scale approach, provided that its secondary structure is known. Starting from a random initial configuration, the folded structure can be obtained in days using a regular desktop computer. Various thermodynamic properties of helix packing of BR are also investigated in this CG model.     

Selectide Technology: Bead-Binding Screening

The Selectide process is a random synthetic chemical library method based on the one-bead one-peptide (structure) concept. A "split-synthesis" method is used to generate huge random libraries (10⁶-10⁸). At the end of the synthesis, each bead expresses only one chemical entity (e.g., peptide). The whole library is then tested simultaneously for binding to a specific acceptor molecule of biologic interest. The ligand bead that interacts specifically with the acceptor molecule is then isolated for structure determination. Once a binding motif is identified, a secondary library (based on the motif of the primary screen) is generated and screened under a more stringent condition to identify leads of higher affinity. This process can be applied to both peptide and nonpeptide (small organic) libraries. In the case of nonsequencable structure libraries, the coding principle has to be applied for structure elucidation of positively reacting beads. Coding peptide is synthesized in parallel to the screening structure, and classical Edman degradation (one or multiple-step) is used for structural analysis. To exclude the possibility of interaction of the macromolecular target (e.g., receptor, enzyme, antibody) with the coding structure, a synthetic technique for segregation of the surface (screening structure) and the interior (coding structure) of the beads was developed. The one-bead one-structure process is invaluable in drug discovery for lead identification as well as further optimization of the initial leads. It also serves as an important research tool for molecular recognition.     

Asymmetric stability among the transmembrane helices of lactose permease

Combining structure determinations from nuclear magnetic resonance (NMR) data and molecular dynamics simulations (MD) under the same environmental conditions revealed a startling asymmetry in the intrinsic conformational stability of secondary structure in the transmembrane domain of lactose permease (LacY). Eleven fragments, corresponding to transmembrane segments (TMs) of LacY, were synthesized, and their secondary structure in solution was determined by NMR. Eight of the TMs contained significant regions of helical structure. MD simulations, both in DMSO and in a DMPC bilayer, showed sites of local stability of helical structure in these TMs, punctuated by regions of conformational instability, in substantial agreement with the NMR data. Mapping the stable regions onto the crystal structure of LacY reveals a marked asymmetry, contrasting with the pseudosymmetry in the static structure: the secondary structure in the C-terminal half is more stable than in the N-terminal half. The relative stability of secondary structure is likely exploited in the transport mechanism of LacY. Residues supporting proton conduction are in more stable regions of secondary structure, while residues key to substrate binding are found in considerably unstable regions of secondary structure.     

Structural dissection of alkaline-denatured pepsin

It has been established in a number of studies that the alkaline-denatured state of pepsin (the I(P) state) is composed of a compact C-terminal lobe and a largely unstructured N-terminal lobe. In the present study, we have investigated the residual structure in the I(P) state in more detail, using limited proteolysis to isolate and characterize a tightly folded core region from this partially denatured pepsin. The isolated core region corresponds to the 141 C-terminal residues of the pepsin molecule, which in the fully native state forms one of the two lobes of the structure. A comparative study using NMR and CD spectroscopy has revealed, however, that the N-terminal lobe contributes a substantial amount of additional residual structure to the I(P) state of pepsin. CD spectra indicate in addition that significant nonnative alpha-helical structure is present in the C-terminal lobe of the structure when the N-terminal lobe of pepsin is either unfolded or removed by proteolysis. This study demonstrates that the structure of pepsin in the I(P) state is significantly more complex than that of a fully folded C-terminal lobe connected to an unstructured N-terminal lobe.     

The solution structure of a monomeric insulin. A two-dimensional 1H-NMR study of des-(B26-B30)-insulin in combination with distance geometry and restrained molecular dynamics.

The solution conformation of des-(B26-B30)-insulin (DPI) has been investigated by 1H-NMR spectroscopy. A set of 250 approximate interproton distance restraints, derived from two-dimensional nuclear Overhauser enhancement spectra, were used as the basis of a structure determination using distance geometry (DG) and distance-bound driven dynamics (DDD). Sixteen DG structures were optimized using energy minimization (EM) and submitted to short 5-ps restrained molecular dynamics (RMD) simulations. A further refinement of the DDD structure with the lowest distance errors was done by energy minimization, a prolonged RMD simulation in vacuo and a time-averaged RMD simulation. An average structure was obtained from a trajectory generated during 20-ps RMD. The final structure was compared with the des-(B26-B30)-insulin crystal structure refined by molecular dynamics and the 2-Zn crystal structure of porcine insulin. This comparison shows that the overall structure of des-(B26-B30)-insulin is retained in solution with respect to the crystal structures with a high flexibility at the N-terminal part of the A chain and at the N-terminal and C-terminal parts of the B chain. In the RMD run a high mobility of Gly A1, Asn A21 and of the side chain of Phe B25 is noticed. One of the conformations adopted by des-(B26-B30)-insulin in solution is similar to that of molecule 1 (Chinese nomenclature) in the crystal structure of porcine insulin.     

Oligoglycine surface structures: molecular dynamics simulation

The full-atomic molecular dynamics (MD) simulation of adsorption mode for diantennary oligoglycines [H-Gly4-NH(CH2)5]2 onto graphite and mica surface is described. The resulting structure of adsorption layers is analyzed. The peptide second structure motives have been studied by both STRIDE (structural identification) and DSSP (dictionary of secondary structure of proteins) methods. The obtained results confirm the possibility of polyglycine II (PGII) structure formation in diantennary oligoglycine (DAOG) monolayers deposited onto graphite surface, which was earlier estimated based on atomic-force microscopy measurements.     

Understanding the roles of amino acid residues in tertiary structure formation of chignolin by using molecular dynamics simulation

Chignolin is a 10-residue peptide (GYDPETGTWG) that forms a stable beta-hairpin structure in water. However, its design template, GPM12 (GYDDATKTFG), does not have a specific structure. To clarify which amino acids give it the ability to form the beta-hairpin structure, we calculated the folding free-energy landscapes of chignolin, GPM12, and their chimeric peptides using multicanonical molecular dynamics (MD) simulation. Cluster analysis of the conformational ensembles revealed that the native structure of chignolin was the lowest in terms of free energy while shallow local minima were widely distributed in the free energy landscape of GPM12, in agreement with experimental observations. Among the chimeric peptides, GPM12(D4P/K7G) stably formed the same beta-hairpin structure as that of chignolin in the MD simulation. This was confirmed by nuclear magnetic resonance (NMR) spectroscopy. A comparison of the free-energy landscapes showed that the conformational distribution of the Asp3-Pro4 sequence was inherently biased in a way that is advantageous both to forming hydrogen bonds with another beta-strand and to initiating loop structure. In addition, Gly7 helps stabilize the loop structure by having a left-handed alpha-helical conformation. Such a conformation is necessary to complete the loop structure, although it is not preferred by other amino acids. Our results suggest that the consistency between the short-range interactions that determine the local geometries and the long-range interactions that determine the global structure is important for stable tertiary structure formation.     

MMDB: Entrez's 3D-structure database

Three-dimensional structures are now known within most protein families and it is likely, when searching a sequence database, that one will identify a homolog of known structure. The goal of Entrez's 3D-structure database is to make structure information and the functional annotation it can provide easily accessible to molecular biologists. To this end, Entrez's search engine provides several powerful features: (i) links between databases, for example between a protein's sequence and structure; (ii) pre-computed sequence and structure neighbors; and (iii) structure and sequence/structure alignment visualization. Here, we focus on a new feature of Entrez's Molecular Modeling Database (MMDB): Graphical summaries of the biological annotation available for each 3D structure, based on the results of automated comparative analysis. MMDB is available at: http://www.ncbi.nlm.nih.gov/Entrez/structure.html.     

抗寒性不同的油橄榄品种和单株叶片扫描电镜观察

抗寒性强的油橄榄——克里302、尼基特371的叶片,扫描电镜下的特征是:(1) 近轴表皮角质膜厚,蜡质结晶致密。(2) 远轴表皮内陷气孔,密被鳞毛。(3) 叶两面栅栏组织发达,为旱生形态阳叶结构。     

Protein conformational transitions coupled to binding in molecular recognition of unstructured proteins: deciphering the effect of intermolecular interactions on computational structure prediction of the p27Kip1 protein bound to the cyclin A-cyclin-dependent kinase 2 complex

The relationship between folding mechanism coupled to binding and structure prediction of the tertiary complexes is studied for the p27(Kip) (1) protein which has an intrinsically disordered unbound form and undergoes a functional folding transition during complex formation with the phosphorylated cyclin A-cyclin-dependent kinase 2 (Cdk2) binary complex. Hierarchy of p27(Kip1) structural loss determined in our earlier studies from temperature-induced Monte Carlo simulations and subsequent characterization of the transition state ensemble (TSE) for the folding reaction have shown that simultaneous ordering of the p27(Kip1) native intermolecular interface for the beta-hairpin and beta-strand secondary structure elements is critical for nucleating a rapid kinetic transition to the native tertiary complex. In the present study, we investigate the effect of forming specific intermolecular interactions on structure prediction of the p27(Kip1) tertiary complex. By constraining different secondary structure elements of p27(Kip1) in their native bound conformations and conducting multiple simulated annealing simulations, we analyze differences in the success rate of predicting the native structure of p27(Kip1) in the tertiary complex. In accordance with the nucleation-condensation mechanism, we have found that further stabilization of the native intermolecular interface for the beta-hairpin and beta-strand elements of p27(Kip1), that become ordered in the TSE, but are hardly populated in the unbound state, results in a consistent acquisition of the native bound structure. Conversely, the excessive stablization of the local secondary structure elements, which are rarely detected in the TSE, has a detrimental effect on convergence to the native bound structure.     

Conformational studies on sequential polypeptides Part VI. Structural investigations on (Pro-Leu-Gly)10, (Pro-Leu-Gly)n and (Leu-Pro-Gly)n.

The conformational properties of (Pro-Leu-Gly)10, (Pro-Leu-Gly)n and (Leu-Pro-Gly)n were investigated both in solution and in solid state. By circular dichroism studies it was possible to demonstrate the formation of an ordered collagen-like structure for (Pro-Leu-Gly)n in hexafluroisopropanol-water mixtures and in ethylene glycol; (Leu-Pro-Gly)n assumes an ordered conformation only in ethylene glycol; (Pro-Leu-Gly)10 is unordered under all the conditions studied. X-ray diffraction patterns indicated that (Pro-Leu-Gly)n and (Leu-Pro-Gly)n assume a triple helical structure in solid state. In addition, the investigation of (Pro-Leu-Gly)n strongly suggests that this type of structure is a single chain triple helix. The X-ray patterns of (Pro-Leu-Gly)10 do not allow us to ascertain a collagen or polyproline II-like structure for this decatripeptide.