Protein Functional Relationships Research Articles

Using the underlying biological organization of the Mycobacterium tuberculosis functional network for protein function prediction

Despite ever-increasing amounts of sequence and functional genomics data, there is still a deficiency of functional annotation for many newly sequenced proteins. For Mycobacterium tuberculosis (MTB), more than half of its genome is still uncharacterized, which hampers the search for new drug targets within the bacterial pathogen and limits our understanding of its pathogenicity. As for many other genomes, the annotations of proteins in the MTB proteome were generally inferred from sequence homology, which is effective but its applicability has limitations. We have carried out large-scale biological data integration to produce an MTB protein functional interaction network. Protein functional relationships were extracted from the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database, and additional functional interactions from microarray, sequence and protein signature data. The confidence level of protein relationships in the additional functional interaction data was evaluated using a dynamic data-driven scoring system. This functional network has been used to predict functions of uncharacterized proteins using Gene Ontology (GO) terms, and the semantic similarity between these terms measured using a state-of-the-art GO similarity metric. To achieve better trade-off between improvement of quality, genomic coverage and scalability, this prediction is done by observing the key principles driving the biological organization of the functional network. This study yields a new functionally characterized MTB strain CDC1551 proteome, consisting of 3804 and 3698 proteins out of 4195 with annotations in terms of the biological process and molecular function ontologies, respectively. These data can contribute to research into the Development of effective anti-tubercular drugs with novel biological mechanisms of action.

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Gaia: automated quality assessment of protein structure models

Increasing use of structural modeling for understanding structure-function relationships in proteins has led to the need to ensure that the protein models being used are of acceptable quality. Quality of a given protein structure can be assessed by comparing various intrinsic structural properties of the protein to those observed in high-resolution protein structures. In this study, we present tools to compare a given structure to high-resolution crystal structures. We assess packing by calculating the total void volume, the percentage of unsatisfied hydrogen bonds, the number of steric clashes and the scaling of the accessible surface area. We assess covalent geometry by determining bond lengths, angles, dihedrals and rotamers. The statistical parameters for the above measures, obtained from high-resolution crystal structures enable us to provide a quality-score that points to specific areas where a given protein structural model needs improvement. We provide these tools that appraise protein structures in the form of a web server Gaia (http://chiron.dokhlab.org). Gaia evaluates the packing and covalent geometry of a given protein structure and provides quantitative comparison of the given structure to high-resolution crystal structures. dokh@unc.edu Supplementary data are available at Bioinformatics online.

Hidden dynamic allostery in a PDZ domain

Structure-function relationships in proteins are predicated on the spatial proximity of noncovalently interacting groups of atoms. Thus, structural elements located away from a protein's active site are typically presumed to serve a stabilizing or scaffolding role for the larger structure. Here we report a functional role for a distal structural element in a PDZ domain, even though it is not required to maintain PDZ structure. The third PDZ domain from PSD-95/SAP90 (PDZ3) has an unusual additional third alpha helix (alpha3) that packs in contiguous fashion against the globular domain. Although alpha3 lies outside the active site and does not make direct contact with C-terminal peptide ligand, removal of alpha3 reduces ligand affinity by 21-fold. Further investigation revealed that the difference in binding free energies between the full-length and truncated constructs is predominantly entropic in nature and that without alpha3, picosecond-nanosecond side-chain dynamics are enhanced throughout the domain, as determined by (2)H methyl NMR relaxation. Thus, the distal modulation of binding function appears to occur via a delocalized conformational entropy mechanism. Without removal of alpha3 and characterization of side-chain dynamics, this dynamic allostery would have gone unnoticed. Moreover, what appeared at first to be an artificial modification of PDZ3 has been corroborated by experimentally verified phosphorylation of alpha3, revealing a tangible biological mechanism for this novel regulatory scheme. This hidden dynamic allostery raises the possibility of as-yet unidentified or untapped allosteric regulation in this PDZ domain and is a very clear example of function arising from dynamics rather than from structure.

Chapter 19 - Predicting pKa Values with Continuous Constant pH Molecular Dynamics

Knowledge of pK(a) values is important for understanding structure and function relationships in proteins. Over the past two decades, theoretical methods for pK(a) calculations have been mainly based on macroscopic models, in which the protein is considered as a low-dielectric cavity embedded in a high-dielectric continuum. In recent years, constant pH molecular dynamics methods have been developed based on a microscopic description of the protein. We describe here the methodology of continuous constant pH molecular dynamics (CPHMD), which has emerged as one of the most robust and accurate tools for predicting protein pK(a)s and for the study of pH-modulated conformational dynamics. We illustrate the utility of CPHMD by the calculation of pK(a)s for surface residues in ribonuclease A, buried residues in staphylococcal nuclease, and titratable groups in the intrinsically flexible protein α-lactalbumin. We will compare the CPHMD results with experimental data as well as calculations from PB-based and empirical methods. These examples demonstrate the accuracy and robustness of the CPHMD method and its ability to capture the correlation between ionization equilibria and conformational dynamics as well as the local dielectric response to structural rearrangement. Finally, we discuss future improvement of the CPHMD method.

Protein co-evolution, co-adaptation and interactions

Co-evolution has an important function in the evolution of species and it is clearly manifested in certain scenarios such as host–parasite and predator–prey interactions, symbiosis and mutualism. The extrapolation of the concepts and methodologies developed for the study of species co-evolution at the molecular level has prompted the development of a variety of computational methods able to predict protein interactions through the characteristics of co-evolution. Particularly successful have been those methods that predict interactions at the genomic level based on the detection of pairs of protein families with similar evolutionary histories (similarity of phylogenetic trees: mirrortree). Future advances in this field will require a better understanding of the molecular basis of the co-evolution of protein families. Thus, it will be important to decipher the molecular mechanisms underlying the similarity observed in phylogenetic trees of interacting proteins, distinguishing direct specific molecular interactions from other general functional constraints. In particular, it will be important to separate the effects of physical interactions within protein complexes (‘co-adaptation') from other forces that, in a less specific way, can also create general patterns of co-evolution.

Determination of helix orientations in proteins

Accurate description of helices, including curvature and bending, and determination of interhelical angles are essential for analysis of the three-dimensional fold and functionally important conformational changes in helical proteins. Here, a new computational method is presented that allows determination of angles between any helical stretches, the radius of curvature of curved helices, bending angle of bent helices, as well as symmetry relations within the protein molecule, using main chain atom coordinates. The method has been applied to describe changes in interhelical angles in calmodulin upon interaction with a target peptide, which reveals the conformational changes at a higher precision. Because subtle changes in helix-to-helix packing and interhelical angles often underlie significant functional transitions in proteins, this approach can serve as a useful tool for characterization of such conformational changes at an exceedingly high accuracy and thus provide detailed insight into the structure–function relationship of proteins.

Chemical rescue of a site-modified ligand to a [4Fe–4S] cluster in PsaC, a bacterial-like dicluster ferredoxin bound to Photosystem I

Chemical rescue of site-modified amino acids using externally supplied organic molecules represents a powerful method to investigate structure–function relationships in proteins. Here we provide definitive evidence that aryl and alkyl thiolates, reagents typically used for in vitro iron–sulfur cluster reconstitutions, serve as rescue ligands to a site-specifically modified [4Fe–4S] 1+,2+ cluster in PsaC, a bacterial dicluster ferredoxin-like subunit of Photosystem I. PsaC binds two low-potential [4Fe–4S] 1+,2+ clusters termed F A and F B. In the C13G/C33S variant of PsaC, glycine has replaced cysteine at position 13 creating a protein that is missing one of the ligating amino acids to iron–sulfur cluster F B. Using a variety of analytical techniques, including non-heme iron and acid-labile sulfur assays, and EPR, resonance Raman, and Mössbauer spectroscopies, we showed that the C13G/C33S variant of PsaC binds two [4Fe–4S] 1+,2+ clusters, despite the absence of one of the biological ligands. 19F NMR spectroscopy indicated that the external thiolate replaces cysteine 13 as a substitute ligand to the F B cluster. The finding that site-modified [4Fe–4S] 1+,2+ clusters can be chemically rescued with external thiolates opens new opportunities for modulating their properties in proteins. In particular, it provides a mechanism to attach an additional electron transfer cofactor to the protein via a bound, external ligand.

Automated high-throughput process for site-directed mutagenesis, production, purification, and kinetic characterization of enzymes

Site-directed mutagenesis followed by functional characterization is a widely used approach to obtain information on the structure–function relationship of proteins. Due to time and cost considerations, the number of amino acids studied is frequently reduced. To address the need for convenient parallel production of numerous point mutants of a protein, we developed an automated method to perform classical site-directed mutagenesis, protein purification, and characterization in a high-throughput manner. The process consists of a succession of six fully automated protocols that can be adapted to any automated liquid handling systems. Our procedure allows construction, validation, and characterization of hundreds of site-directed mutants of a given protein in just 4 days. The method is especially adapted to projects aiming at the study of unique or multiple mutants without the need to construct and screen large libraries of random mutants. The usefulness of the technique is illustrated by the construction and characterization of tens of single mutants of the penicillin-binding protein 2x (PBP2x) from Streptococcus pneumoniae. Moreover, seven mutations of PBP2x were obtained simultaneously in a single experiment with efficiency close to 90%.

Application of FTTP to alpha-helix or beta-strand motifs

Information concerning protein structure is widely dispersed and cannot easily and rapidly be processed by the biological community. We present a database of tendentious factors of three states of tripeptide units from PDB database, called a bank of tendentious factors of three states of three-peptide units (FTTP). The FTTP database was constructed based on conformational dihedral angle ( ϕ, ψ) library of 20 3 peptide triplets by exhaustively searching through PDB databases. We introduce the FTTP database for the analysis of characteristics common to relative conformational biases of all peptide triplets, especially finding some motifs apt to alpha-helix and beta-strand. Our results show that this will provide a platform for studies of short peptide motifs, folding codons, secondary structure and three-dimensional (3D) structure of proteins. Moreover, FTTP is a unique resource that will allow a comprehensive characterization of peptide triplets and thus improve our understanding of sequence–structure relationship, refined domains, 3D structures, and their associated function. We believe the FTTP database will help biologists in increasing the efficiency of finding useful and relevant information regarding structure–function relationship of proteins. Therefore, this approach will play an important role in protein folding, protein engineering, molecular design, and proteomics.

Detection of Activity Centers in Cellular Pathways Using Transcript Profiling

We present a new computational method for identifying regulated pathway components in transcript profiling (TP) experiments by evaluating transcriptional activity in the context of known biological pathways. We construct a graph representing thousands of protein functional relationships by integrating knowledge from public databases and review articles. We use the notion of distance in a graph to define pathway neighborhoods. The pathways perturbed in an experiment are then identified as the subgraph induced by the genes, referred to as activity centers, having significant density of transcriptional activity in their functional neighborhoods. We illustrate the predictive power of this approach by performing and analyzing an experiment of TP53 overexpression in NCI-H125 cells. The detected activity centers are in agreement with the known TP53 activation effects and our independent experimental results. We also apply the method to a serum starvation experiment using HEY cells and investigate the predicted activity of the transcription factor MYC. Finally, we discuss interesting properties of the activity center approach and its possible applications beyond the comparison of two experiments.

Non-random base composition in codons of mitochondrial cytochrome b gene in vertebrates.

Cytochrome b is the central catalytic subunit of the quinol:cytochrome c oxidoreductase of complex III of the mitochondrial oxidative phosphorylation system and is essential to the viability of most eukaryotic cells. Partial cytochrome b gene sequences of 14 species representing mammals, birds, reptiles and amphibians are presented here including some species typical for Poland. For the analysed species a comparative analysis of the natural variation in the gene was performed. This information has been used to discuss some aspects of gene sequence - protein function relationships. Review of relevant literature indicates that similar comparisons have been made only for basic mammalian species. Moreover, there is little information about the Polish-specific species. We observed that there is a strong non-random distribution of nucleotides in the cytochrome b sequence in all tested species with the highest differences at the third codon position. This is also the codon position of the strongest compositional bias. Some tested species, representing distant systematic groups, showed unique base composition differing from the others. The quail, frog, python and elk prefer C over A in the light DNA strand. Species belonging to the artiodactyls stand out from the remaining ones and contain fewer pyrimidines. The observed overall rate of amino acid identity is about 61%. The region covering Q(o) center as well as histidines 82 and 96 (heme ligands) are totally conserved in all tested species. Additionally, the applied method and the sequences can also be used for diagnostic species identification by veterinary and conservation agencies.

Pullulan Degrading Enzymes of Bacterial Origin

Pullulan degrading enzymes belong to a group of glycosylhydrolases that are widely distributed in nature and are produced by an extremely wide variety of species. Among them the thermophilic and mesophilic bacteria are a rich source of these enzymes. There are many biotechnological applications for these enzymes and a rapidly growing amount of information about their diversity, genetic as well as biochemical and biophysical characteristics. The properties of these enzymes vary and are somewhat linked to the natural environment inhabited by the producing organisms. Genes for these enzymes have been cloned from several strains and their amino acid sequences show highly conserved regions common to the enzymes of the amylase family. Molecular studies have greatly extended our knowledge on pullulan degrading enzymes and their biosynthesis. However, enzyme production levels have usually not been as high as had been assumed possible, and the properties of some enzymes are less than optimal for their industrial applications. Some of these problems can be overcome with the use of good producer organisms, optimized expression/secretion vectors, and site-directed mutagenesis. The molecular biology of pullulan degrading enzymes has been and continues to be a valuable system for studying basic questions of cell biology, such as mechanisms of gene regulation and secretion, and the structure–function relationships of proteins.

Multiple site-directed mutagenesis of more than 10 sites simultaneously and in a single round

Site-directed mutagenesis is a powerful tool to explore the structure–function relationship of proteins, but most traditional methods rely on the mutation of only one site at a time and efficiencies drop drastically when more than three sites are targeted simultaneously. Many applications in functional proteomics and genetic engineering, including codon optimization for heterologous expression, generation of cysteine-less proteins, and alanine-scanning mutagenesis, would greatly benefit from a multiple-site mutagenesis method with high efficiency. Here we describe the development of a simple and rapid method for site-directed mutagenesis of more than 10 sites simultaneously with up to 100% efficiency. The method uses two terminal tailed primers with a unique 25-nucleotide tail each that are simultaneously annealed to template DNA together with the set of mutagenic primers inbetween. Following synthesis of the mutant strand by primer extension and ligation with T4 DNA polymerase and ligase, the unique mutant strand-specific tails of the terminal primers are used as anchors to specifically amplify the mutant strand by high-fidelity polymerase chain reaction. We have employed this novel method to mutate simultaneously all 9 and 11 CUG leucine codons of the Hyg and Neo resistance genes, respectively, to the Candida albicans-friendly UUG leucine codon at 100% efficiency.

Rapid, high-throughput purification of HIV-1 integrase using microtiter plate technology

The human immunodeficiency virus type-1 (HIV-1) integrase (IN) catalyzes the insertion of the retroviral genome into the chromosome of an infected host cell. HIV-1 IN was expressed as a N-terminal hexa-histidine fusion in Escherichia coli. A high-throughput purification strategy was developed using denaturing methods for the initial protein extraction, followed by a one-step nickel-chelating chromatography purification and step-wise refolding. IN was routinely greater than 90% pure with yields exceeding 14 μg of purified IN per ml of E. coli culture. In vitro 3 ′ processing and strand transfer assays showed the enzyme preparations to be highly active. The specific activity of the purified IN was 2.65 pmol/h/μg IN, which is very similar to the activity of IN routinely produced by large-scale column chromatographic methods. This high-throughput platform should be of general utility to those interested in defining the structure–function relationship of proteins and enzymes.

Protein-protein and protein-function relationships in Arabidopsis photosystem I: cluster analysis of PSI polypeptide levels and photosynthetic parameters in PSI mutants

In flowering plants, photosystem I (PSI) mediates electron transport across the thylakoid membrane and contains at least 14 proteins. The availability of co-suppression and/or mutant lines deficient for individual PSI polypeptides in Arabidopsis thaliana allows one to assign functions to PSI subunits. We have performed cluster analysis on an extensive set of data on PSI polypeptide levels in ten different PSI mutants. This type of analysis serves to group proteins that exhibit similar changes in amount in different genotypes, and also identifies genotypes which show similar PSI compositions. The interdependence of levels of PSI-C, -D and -E, of -H and -L, and of Lhca2 and 3, which was previously proposed based on the study of single genotypes or on cross-linking experiments, was confirmed by our analyses. In addition, the levels of the lumenal subunits F and N are found to be interdependent. The incorporation of photosynthetic parameters into the cluster analysis revealed that the level of photosynthetic state transitions correlates with the abundance of PSI-H in all 8 genotypes tested, supporting the hypothesis that PSI-H serves as a docking site for LHCII during state transitions.

Structure and function relationships in proteins: a tribute to Allen B. Edmundson

Journal of Molecular RecognitionVolume 15, Issue 5 p. 225-226 EditorialFree Access Structure and function relationships in proteins: a tribute to Allen B. Edmundson† Paul A. Ramsland, Corresponding Author Paul A. Ramsland Guest Editor [email protected] Structural Biology Laboratory, The Austin Research Institute, Victoria, AustraliaStructural Biology Laboratory, The Austin Research Institute, Studley Road, Heidelberg, Victoria 3084, AustraliaSearch for more papers by this author Paul A. Ramsland, Corresponding Author Paul A. Ramsland Guest Editor [email protected] Structural Biology Laboratory, The Austin Research Institute, Victoria, AustraliaStructural Biology Laboratory, The Austin Research Institute, Studley Road, Heidelberg, Victoria 3084, AustraliaSearch for more papers by this author First published: 19 November 2002 https://doi.org/10.1002/jmr.582 † This paper is published as part of a special issue entitled “Structure and function relationships in proteins: a tribute to Allen B. Edmundson”. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. REFERENCES Edmundson AB. 1963. Cleavage of sperm whale myoglobin with cyanogen bromide. Nature 198: 354– 357. Edmundson AB. 1965. Amino-acid sequence of sperm whale myoglobin. Nature 205: 883– 887. Edmundson AB, Hirs CHW. 1961. The amino-acid sequence of sperm whale myoglobin. Nature 190: 663– 672. Edmundson AB, Ely KR, Girling RL, Abola EE, Schiffer M, Westholm FA. 1974a. Structure and binding properties of a λ-type Bence–Jones dimer. In Progress in Immunology II, L Brent, J Holborow (eds), Vol. 1. North-Holland: Amsterdam; 103– 113. Edmundson AB, Ely KR, Girling RL, Abola EE, Schiffer M, Westholm FA, Fausch MD, Deutsch HF. 1974b. Binding of 2,4-dinitrophenyl compounds and other small molecules to a crystalline λ-type Bence–Jones dimer. Biochemistry 13: 3816– 3827. Edmundson AB, Ely KR, Abola EE, Schiffer M, Panagiotopoulos N. 1975. Rotational allomerism and divergent evolution of domains in immunoglobulin light chains. Biochemistry 14: 3953– 3961. Kendrew JC, Watson HC, Strandberg BE, Dickerson RE, Phillips DC, Shore VC. 1961. Structure of myoglobin. A partial determination by X-ray methods and its correlation with chemical data. Nature 190: 666– 670. Koshland DE Jr. 1973. Protein and biological control. Sci. Am. 229: 52– 64. Schiffer M, Edmundson AB. 1967. Use of helical wheels to represent the structures of proteins and to identify segments with helical potential. Biophys. J. 7: 121– 135. Schiffer M, Girling RL, Ely KR, Edmundson AB. 1973. Structure of a λ-type Bence–Jones protein at 3.5 Å resolution. Biochemistry 12: 4620– 4631. Watson HC, Kendrew JC. 1961. Comparison between the amino-acid sequences of sperm whale myoglobin and human myoglobin. Nature 190: 670– 672. Volume15, Issue5Special Issue: Structure and Function Relationships in Proteins: A Tribute to Allen B. EdmundsonSeptember/October 2002Pages 225-226 ReferencesRelatedInformation

Characterization of the folding degree of proteins.

The characterization of the folding degree of chains is central to the elucidation of structure--function relationships in proteins. Here we present a new index for characterizing the folding degree of a (protein) chain. This index shows a range of features that are desirable for the study of the relation between structure and function in proteins. A novel index characterizing the folding degree of (protein) chains is developed based on the spectral moments of a matrix representing the dihedral angles (phi, omega and epsilon) of the protein main chain. The proposed index is normalized to the chain size, is not correlated to the gyration radius of the backbone chain and is able to distinguish between structures for which the sum of the main-chain dihedral angles is identical. The index is well correlated to the percentages of helix and strand in proteins, shows a linear dependence with temperature changes, and is able to differentiate among protein families. On request from the author.

Interactions in Plastocyanin−Lysine Peptide and Related Systems

The structure−function relationship of proteins is one of the major topics of protein studies. In spite of the importance of intermolecular interactions between proteins, which could control their functions, there are only a limited number of detailed studies on the protein structural changes induced by interaction with a protein or a peptide, because the associated changes in properties are small. Charged peptides, however, may be useful for studying the molecular recognition character of proteins and their interaction-induced structural changes, since they do not have any visible absorption. The active site Cu of plastocyanin (PC) exhibited a longer Cu−S(Cys) bond length and a higher redox potential on binding of a lysine peptide, suggesting that lysine peptides induce a structural change in PC to adapt the copper site for facile electron transfer. Structural changes were also observed for cytochrome (cyt) f, cyt c, and cytochrome c peroxidase on interaction with charged peptides.

Characterize Protein Functional Relationships Based on Mrna Expression Profile.

INTRODUCTION. Protein families are distinguished by members that exhibit sequence and biomedical function similarity. For most gene changes in protein abundance are related to changes in mRNA abundance, which are immensely informative about cell state and the activity of genes. LifeExpress RNA (LE) database (Incyte Genomics, Inc) is a large-scale genome expression database. Based on LE we derived the functional relationship of Pfam[1], a database of protein domain families, by studying the global expression profile of the corresponding genes of Pfam family members. The expression profiles for 135 largest Pfam families were summarized and relationships were analyzed. The study present a simple model for conceptualizing the complex genetic regulatory network.