Detection of discriminative sequence motifs in proteins obtained from prokaryotes grown at various temperatures

Recent investigations on the stability of proteins have demonstrated various structural factors, but few have considered sequence factors such as protein motifs. These motifs represent highly conserved regions and describe critical regions that may only exist on proteins that remain functional at high temperatures. This investigation presents a method for identifying and comparing corresponding mesophilic and thermophilic sequence motifs between protein families. Discriminative motifs that are conserved only in the mesophilic or thermophilic subfamily are identified. Analysis of the results shows that, although the subfamilies of most protein families share similar motifs, some discriminative motifs are present in particular thermophilic/mesophilic subfamilies. The thermophilic discriminative motifs are conserved only in thermophilic organisms, revealing that physiochemical principles support thermostability.     

Phosphate recognition in structural biology

Drug-discovery research in the past decade has seen an increased selection of targets with phosphate recognition sites, such as protein kinases and phosphatases, in the past decade. This review attempts, with the help of database-mining tools, to give an overview of the most important principles in molecular recognition of phosphate groups by enzymes. A total of 3003 X-ray crystal structures from the RCSB Protein Data Bank with bound organophosphates has been analyzed individually, in particular for H-bonding interactions between proteins and ligands. The various known binding motifs for phosphate binding are reviewed, and similarities to phosphate complexation by synthetic receptors are highlighted. An analysis of the propensities of amino acids in various classes of phosphate-binding enzymes showed characteristic distributions of amino acids used for phosphate binding. This review demonstrates that structure-based lead development and optimization should carefully address the phosphate-binding-site environment and also proposes new alternatives for filling such sites.     

A data mining and ab initio study of the interaction between the aromatic and backbone amide groups in proteins

Interactions between aromatic groups and backbone amide groups in protein environments are characterized both through data mining analyses of X‐ray protein structures and through ab initio molecular orbital calculations on a model complex. The data mining analyses of 1029 X‐ray protein structures elucidate the configurational characteristics of the interaction as well as the positions of the interacting moieties involved. On a statistical average, more than seven such interactions occur in a typical protein structure. The configurations of these interactions are restricted with the face‐to‐face orientation as the preferred arrangement. The interaction occurs mainly within a single peptide chain. Both α‐helix and β‐strand secondary structures provide an almost equal number of backbone amides to participate within this interaction. The interaction energy was evaluated with the supermolecular ab initio method at the MP2 level. It is shown that aromatic–amide(backbone) interactions identified in proteins can achieve a stabilization energy of 3.3 kcal/mol. The interaction involves the entirety of the backbone amide rather than only its amine portion. This study concludes that the interaction between aromatic and backbone amide groups is of general significance to protein structure due to its strength and common occurrence. © 2000 John Wiley & Sons, Inc. Int J Quant Chem 80: 44–60, 2000     

Detecting conserved secondary structures in RNA molecules using constrained structural alignment

Constrained sequence alignment has been studied extensively in the past. Different forms of constraints have been investigated, where a constraint can be a subsequence, a regular expression, or a probability matrix of symbols and positions. However, constrained structural alignment has been investigated to a much lesser extent. In this paper, we present an efficient method for constrained structural alignment and apply the method to detecting conserved secondary structures, or structural motifs, in a set of RNA molecules. The proposed method combines both sequence and structural information of RNAs to find an optimal local alignment between two RNA secondary structures, one of which is a query and the other is a subject structure in the given set. The method allows a biologist to annotate conserved regions, or constraints, in the query RNA structure and incorporates these regions into the alignment process to obtain biologically more meaningful alignment scores. A statistical measure is developed to assess the significance of the scores. Experimental results based on detecting internal ribosome entry sites in the RNA molecules of hepatitis C virus and Trypanosoma brucei demonstrate the effectiveness of the proposed method and its superiority over existing techniques.     

Dynamic consequences of mutating the typical HPGG motif of apocytochrome b_5 revealed by computer simulation

Apecytochrome b5 with a typical heme-binding motif of HPGC,and its variants with mutated motifs,GPGG,GPGH,HVGG,and HPGP,have been subjected to molecular dynamics simulation.Comparison of the dynamic consequences has revealed the crucial role of HPGG in assembling the heme group of cytochrome b5 and in modulating protein structure,property and function.     

Automatic knowledge extraction from chemical structures: the case of mutagenicity prediction

This work proposes a new structure–activity relationship (SAR) approach to mine molecular fragments that act as structural alerts for biological activity. The entire process is designed to fit with human reasoning, not only to make the predictions more reliable but also to permit clear control by the user in order to meet customized requirements. This approach has been tested on the mutagenicity endpoint, showing marked prediction skills and, more interestingly, bringing to the surface much of the knowledge already collected in the literature as well as new evidence.     

Chemical cross-linking and mass spectrometry for mapping three-dimensional structures of proteins and protein complexes

Chemical cross-linking of proteins, an established method in protein chemistry, has gained renewed interest in combination with mass spectrometric analysis of the reaction products for elucidating low-resolution three-dimensional protein structures and interacting sequences in protein complexes. The identification of the large number of cross-linking sites from the complex mixtures generated by chemical cross-linking, however, remains a challenging task. This review describes the most popular cross-linking reagents for protein structure analysis and gives an overview of the strategies employing intra- or intermolecular chemical cross-linking and mass spectrometry. The various approaches described in the literature to facilitate detection of cross-linking products and also computer software for data analysis are reviewed. Cross-linking techniques combined with mass spectrometry and bioinformatic methods have the potential to provide the basis for an efficient structural characterization of proteins and protein complexes.     

A biomimetic approach to the synthesis of a mycolic acid motif

A new method for the stereoselective synthesis of the (R,R)-β-hydroxy-α-alkyl fatty acid fragment of mycolic acids, via an asymmetric anti-aldol reaction is reported. The ‘mycolic acid motif’ fragment was prepared in three steps and >98% ee.     

Predicting 15N amide chemical shifts in proteins. I. An additive model for the backbone contribution

Because proteins adopt unique structures, chemically identical nuclei in proteins exhibit different chemical shifts. Amide ¹⁵N chemical shifts have been shown to vary over 20 ppm. The cause of these chemical shift inequivalencies is the different intra‐ and intermolecular interactions that individual nuclei experience at different locations in the protein structure. These chemical shift inequivalencies can be described as structural shifts, the difference between the actual chemical shift and the random coil chemical shift. As a first step toward the prediction of these amide ¹⁵N structural shifts, calculations have been carried out on acetyl‐glycine‐methyl amide to examine how a neighboring peptide group influences the amide ¹⁵N structural shifts. The ϕ,ψ dihedral angle space is completely surveyed, while all other geometrical variables are held fixed, to isolate the effect of the backbone conformation. Similar calculations for a limited number of conformations of acetyl‐glycine‐glycine‐methyl amide were carried out, where the effects of the two terminal peptide groups on the central amide ¹⁵N structural shift are examined. It is shown that the effect of the two adjacent groups can be accurately modeled by combining their individual effects additively. This provides a quite simple method to predict the backbone influence on amide ¹⁵N structural shifts in proteins. © 2001 John Wiley & Sons, Inc. J Comput Chem 22: 366–372, 2001     

Melting domain in proteins

Recent progress in thermodynamic aspects of proteins, free or immobilized on solid support, are described. In agreement with results observed with Ribonuclease A [9], DSC analysis on α-chymotrypsin confirms a decoupling of melting domains with the immobilized protein in a large range ofpH.     

Indexing and mapping of proteins using a modified nonlinear Sammon projection

A modified Sammon algorithm was developed to display a relationship between proteins based on their amino acid composition. In the first stage of the method, a 19-dimensional compositional space of representative proteins was mapped into a two-dimensional space (2D) using the original Sammon projection creating a contour map. In the second stage, this contour map was used as a reference for new proteins projected into 2D. Data analysis showed that proteins belonging to the same structural classes formed characteristic and distinct clusters, which could be potentially useful in the prediction of protein structural classes. However, we observed significant overlapping of the clusters, which may explain the limited success of previous protein folding prediction based solely on amino acid composition. Regardless, the modified Sammon projections can generate a unique index for each individually projected protein related to its amino acid composition, which may be a useful tool in the exploratory classification of proteins. ©1999 John Wiley & Sons, Inc. J Comput Chem 20: 1049–1059, 1999     

Property correlations for chlorofluoro carbons and alternatives using structural parameters

Because of their wide ranging applications, the properties of chlorofluoro carbons (CFCs) and their alternatives are studied and correlated in terms of the parameters derived from molecular structures. The properties correlated are of physical, thermodynamic and transport in nature. The input parameters include molar refraction (R _M), molecular connectivity (χ) and van der Waals equation coefficients (a and b), all of which are easily estimated from molecular structures. The correlations developed for the 11 properties studied showed average absolute deviations ranging between 3.5% and 13.7%. The correlation coefficients calculated for the equations developed ranged between 0.9557 and 0.9921.     

An Inside Job: Molecular Determinants for Postsynaptic Localization of Nicotinic Acetylcholine Receptors

Nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic transmission at neuromuscular and autonomic ganglionic synapses in the peripheral nervous system. The postsynaptic localization of muscle ((α1)₂β1γδ) and neuronal ((α3β4)₂β4) nicotinic receptors at these synapses is mediated by interactions between the nAChR intracellular domains and cytoplasmic scaffolding proteins. Recent high resolution structures and functional studies provide new insights into the molecular determinants that mediate these interactions. Surprisingly, they reveal that the muscle nAChR binds 1–3 rapsyn scaffolding molecules, which dimerize and thereby form an interconnected lattice between receptors. Moreover, rapsyn binds two distinct sites on the nAChR subunit cytoplasmic loops; the MA-helix on one or more subunits and a motif specific to the β subunit. Binding at the latter site is regulated by agrin-induced phosphorylation of βY390, and increases the stoichiometry of rapsyn/AChR complexes. Similarly, the neuronal nAChR may be localized at ganglionic synapses by phosphorylation-dependent interactions with 14-3-3 adaptor proteins which bind specific motifs in each of the α3 subunit cytoplasmic loops. Thus, postsynaptic localization of nAChRs is mediated by regulated interactions with multiple scaffolding molecules, and the stoichiometry of these complexes likely helps regulate the number, density, and stability of receptors at the synapse.     

Sequence classification of water channels and related proteins in view of functional predictions

We have worked with a classification method based upon a notion of probabilistic similarity or “likelihood of similarity” between aligned sequences. One important parameter, among others, affecting the sequence similarities and hence the classification results is the amino acid similarity matrix. We present a method for choosing the most adapted matrix to classify protein sequences. This method has been applied to the transmembrane channels of the major intrinsic protein (MIP) family. At present, two functional subgroups have been well characterized in this family: (1) specific water transport by the aquaporins and (2) small neutral solutes transport. The aim of the present study is to show the usefulness of the classification method in the prediction of sequence segments important for substrate selectivity. Moreover, we show that this method can also be used to predict the function of undetermined MIP proteins. The method could be applied to other protein families as well. Received: 24 April 1998 / Accepted: 4 August 1998 / Published online: 2 November 1998     

蛋白质组衍生肽段文库用于鉴定酪蛋白激酶2的底物模体

模体是蛋白质二级结构上的一种特征序列,激酶底物通常具有一类特征性的模体,识别底物模体对鉴定激酶底物具有重要意义。为了快速鉴定激酶底物模体,将全细胞蛋白质酶解液作为肽段文库的来源,采用碱性磷酸酶去除肽段的固有磷酸化后构建了用于筛选激酶底物模体的肽段文库。该肽段文库是大量非磷酸化肽段的混合物,将此混合肽段与酪蛋白激酶2和三磷酸腺苷作用30 min后,通过固定化金属离子亲和色谱法富集磷酸化肽段,采用反相液相色谱-串联质谱分析,成功地鉴定到472条非冗余底物肽段,包含451个非冗余磷酸化位点,并由此得到底物模体S/T-D/E-x-D/E。该法能够快速准确地筛选出激酶底物模体,对研究激酶-底物识别以及信号转导过程具有重要意义。     

Micelle‐Triggered β‐Hairpin to α‐Helix Transition in a 14‐Residue Peptide from a Choline‐Binding Repeat of the Pneumococcal Autolysin LytA

Choline‐binding modules (CBMs) have a ββ‐solenoid structure composed of choline‐binding repeats (CBR), which consist of a β‐hairpin followed by a short linker. To find minimal peptides that are able to maintain the CBR native structure and to evaluate their remaining choline‐binding ability, we have analysed the third β‐hairpin of the CBM from the pneumococcal LytA autolysin. Circular dichroism and NMR data reveal that this peptide forms a highly stable native‐like β‐hairpin both in aqueous solution and in the presence of trifluoroethanol, but, strikingly, the peptide structure is a stable amphipathic α‐helix in both zwitterionic (dodecylphosphocholine) and anionic (sodium dodecylsulfate) detergent micelles, as well as in small unilamellar vesicles. This β‐hairpin to α‐helix conversion is reversible. Given that the β‐hairpin and α‐helix differ greatly in the distribution of hydrophobic and hydrophilic side chains, we propose that the amphipathicity is a requirement for a peptide structure to interact and to be stable in micelles or lipid vesicles. To our knowledge, this “chameleonic” behaviour is the only described case of a micelle‐induced structural transition between two ordered peptide structures.