Dynamics of Fusarium solani cutinase investigated through structural comparison among different crystal forms of its variants

In characterizing mutants and covalently inhibited complexes of Fusarium solani cutinase, which is a 197-residue lipolytic enzyme, 34 variant structures, crystallizing in 8 different crystal forms, have been determined, mostly at high resolution. Taking advantage of this considerable body of information, a structural comparative analysis was carried out to investigate the dynamics of cutinase. Surface loops were identified as the major flexible protein regions, particularly those forming the active-site groove, whereas the elements constituting the protein scaffold were found to retain the same conformation in all the cutinase variants studied. Flexibility turned out to be correlated with thermal motion. With a given crystal packing environment, a high flexibility turned out to be correlated with a low involvement in crystal packing contacts. The high degree of crystal polymorphism, which allowed different conformations with similar energy to be detected, made it possible to identify motions which would have remained unidentified if only a single crystal form had been available. Fairly good agreement was found to exist between the data obtained from the structural comparison and those from a molecular dynamics (MD) simulation carried out on the native enzyme. The crystallographic approach used in this study turned out to be a suitable tool for investigating cutinase dynamics. Because of the availability of a set of closely related proteins in different crystal environments, the intrinsic drawback of a crystallographic approach was bypassed. By combining several static pictures, the dynamics of the protein could be monitored much more realistically than what can be achieved on the basis of static pictures alone. Proteins 26:442–458 © 1996 Wiley-Liss, Inc.     

Secretion systems and membrane-associated structures in rust fungi after high pressure freezing and freeze-fracturing

Excellent preservation and new structural details can be demonstrated in rust-infected leaf tissue after high pressure freezing and freeze-fracturing. A tubular-vesicular complex was the most remarkable cytoplasmic structure observed in cells of the bean rust fungus Uromyces appendiculatus var. appendiculatus during its establishement in its host Phaseolus vulgaris. In fungal cells undergoing intensive synthesis of wall material, this membranous system extended throughout the cytoplasm; in addition, vesicles were accumulated adjacent to the plasma membrane. Here, membrane-associated configurations were observed which seem to be involved in exo- and/or endocytotic processes. It is assumed that the tubular-vesicular complex belongs to the endomembraneous system of the bean rust fungus and that it is involved in the synthesis and secretion of wall material.     

基于PDB数据库的三个RNA二级结构预测软件评估

随着21世纪分子生物学研究的蓬勃发展,RNA二级结构预测成为其中一项重要内容。由于RNA二级结构预测的准确性最为关键,因此寻找高精度且易操作的二级结构预测工具显得非常重要。本文选取三种简单且易操作的二级结构预测软件,先基于PDB数据库收录的318个RNA发夹序列进行二级结构预测,进而通过比较预测结果与实验测定结果进行软件预测性能评估。比较结果显示,RNAstructure为三个软件中性能最优的RNA二级结构预测软件。     

Precision Glycoproteomics Reveals Distinctive N-Glycosylation in Human Spermatozoa

Spermatozoon represents a very special cell type in human body, and glycosylation plays essential roles in its whole life including spermatogenesis, maturation, capacitation, sperm–egg recognition, and fertilization. In this study, by mapping the most comprehensive N-glycoproteome of human spermatozoa using our recently developed site-specific glycoproteomic approaches, we show that spermatozoa contain a number of distinctive glycoproteins, which are mainly involved in spermatogenesis, acrosome reaction and sperm:oocyte membrane binding, and fertilization. Heavy fucosylation is observed on 14 glycoproteins mostly located at extracellular and cell surface regions in spermatozoa but not in other tissues. Sialylation and Lewis epitopes are enriched in the biological process of immune response in spermatozoa, while bisected core structures and LacdiNAc structures are highly expressed in acrosome. These data deepen our knowledge about glycosylation in spermatozoa and lay the foundation for functional study of glycosylation and glycan structures in male infertility.     

The mechanism of aconitase: 1.8 A resolution crystal structure of the S642a:citrate complex

The crystal structure of the S642A mutant of mitochondrial aconitase (mAc) with citrate bound has been determined at 1.8 A resolution and 100 K to capture this binding mode of substrates to the native enzyme. The 2.0 A resolution, 100 K crystal structure of the S642A mutant with isocitrate binding provides a control, showing that the Ser --> Ala replacement does not alter the binding of substrates in the active site. The aconitase mechanism requires that the intermediate product, cis-aconitate, flip over by 180 degrees about the C alpha-C beta double bond. Only one of these two alternative modes of binding, that of the isocitrate mode, has been previously visualized. Now, however, the structure revealing the citrate mode of binding provides direct support for the proposed enzyme mechanism.     

Analytical Background and Discussion of the Chaperone Model of Prion Diseases

It is generally accepted that prion infection is due solely to a protein i.e. the protein-only hypothesis. The essential constituent of infectious prions is the scrapie prion protein (PrP^Sc) which is chemically indistinguishable from the normal, cellular protein (PrP^C) but exhibits distinct secondary and tertiary structure. This very unusual feature seems to be in contradiction with a major paradigm of present structural biology stated by Anfinsen: a protein folds to the most stable conformation, this means only one structure.In order to reconcile the results obtained on prions with the biophysics of protein folding, a model is proposed. It is based on the hypothesis that a thermodynamically irreversible step is involved in protein folding. The model is then extended to chaperone-assisted protein folding. It is shown that, under certain conditions, the transitory secondary structure formed during the earlier step of folding could interact with chaperone. Analysis shows that chaperone may help the protein to find correct conformation. On the other hand, analysis reveals the possibility that more than one structure may form from a single polypeptide chain. Under these conditions, the behaviour of chaperones resembles the characteristics of prion diseases.     

Relationship Between Amino Acid Properties and Protein Stability: Buried Mutations

In order to understand the mechanism of protein stability and to develop a simple method for predicting mutation-induced stability changes, we analyzed the relationship between stability changes caused by buried mutations and changes in 48 amino acid properties. As expected from the importance of hydrophobicity, properties reflecting hydrophobicity are strongly correlated with the stability of proteins. We found that subgroup classification based on secondary structure increased correlations significantly, and mutations within -strand segments correlated better than did those in -helical segments, which may result from stronger hydrophobicity of the -strands. Multiple regression analyses incorporating combinations of three properties from among all possible combinations of the 48 properties increased the correlation coefficient to 0.88 and by an average of 13% for all data sets. Analyzing the stability of tryptophan synthase mutants with Glu49 replaced by all other residues except Arg revealed that combining buriedness, solvent-accessible surface area for denatured protein, and unfolding Gibbs free energy change increased the correlation to 0.95. Consideration of sequence and structural information (neighboring residues in sequence and in space) did not significantly strengthen the correlations in buried mutations, suggesting that nonspecific interactions dominate in the interior of proteins.     

The Amino Acid Sequence of Ribitol Dehydrogenase-F, a Mutant Enzyme with Improved Xylitol Dehydrogenase Activity

A mutant ribitol dehydrogenase (RDH-F) was purified from Klebsiella aerogenes strain F which evolved from the wild-type strain A under selective pressure to improve growth on xylitol, a poor substrate used as sole carbon source. The ratio of activities on xylitol (500 mM) and ribitol (50 mM) was 0.154 for RDH-F compared to 0.033 for the wild-type (RDH-A) enzyme. The complete amino acid sequence of RDH-F showed the mutations. Q60 for E60 and V215 for L215 in the single polypeptide chain of 249 amino acid residues. Structural modeling based on homologies with two other microbial dehydrogenases suggests that E60 Q60 is a neutral mutation, since it lies in a region far from the catalytic site and should not cause structural perturbations. In contrast, L215 V215 lies in variable region II and would shift a loop that interacts with the NADH cofactor. Another improved ribitol dehydrogenase, RDH-D, contains an A196 P196 mutation that would disrupt a surface -helix in region II. Hence conformational changes in this region appear to be responsible for the improved xylitol specificity.     

Uniform and Residue-specific 15N-labeling of Proteins on a Highly Deuterated Background

A general method for stable-isotope labeling of large proteins is introduced and applied for studies of the E. coli GroE chaperone proteins by solution NMR. In addition to enabling the residue-specific (15)N-labeling of proteins on a highly deuterated background, it is also an efficient approach for uniform labeling. The method meets the requirements of high-level deuteration, minimal cross-labeling and high protein yield, which are crucial for NMR studies of structures with sizes above 150 kDa. The results obtained with the new protocol are compared to other strategies for protein labeling, and evaluated with regard to the influence of external factors on the resulting isotope labeling patterns. Applications with the GroE system show that these strategies are efficient tools for studies of structure, dynamics and intermolecular interactions in large supramolecular complexes, when combined with TROSY- and CRINEPT-based experimental NMR schemes.     

Multiple co-inertia analysis: a tool for assessing synchrony in the temporal variability of aquatic communities

Multitable techniques are rarely used for investigating patterns in ecological data surveys despite their ability to deal with the spatial and/or temporal stability of assemblages. Based on a covariance optimisation criterion, Multiple Co-inertia analysis (MCOA) enables the simultaneous ordination of several tables. Such analysis allows the representation of the stable vs. unstable part of the assemblage structure in comparison to a reference derived from each table. We used MCOA on multiple time series of invertebrate sampling to show that synchrony in the temporal variability of communities can establish between geographically distant locations despite the spatial and temporal plasticity of the faunistic responses to long-term changes in environmental conditions.