Thermostability of Rhodopseudomonas viridis and Rhodospirillum rubrum chromatophores reflecting physiological conditions.

Relationships between growth conditions and thermostability were examined for photosynthetic inner membranes (chromatophores) from Rhodopseudomonas viridis and Rhodospirillum rubrum of which morphology, lipid composition, and protein/lipid rate are rather mutually different. Signals observed by differential scanning calorimetry of the chromatophores were correlated with thermal state transitions of the membrane components by reference to temperature dependencies of circular dichroism and absorption spectra of the purified supramolecule comprising a photoreaction center and surrounding light-harvesting pigment-protein complexes that are the prominent proteins in both membranes. The differential scanning calorimetry curves of those chromatophores exhibited different dependencies on growth stages and environmental temperatures.

ProTherm and ProNIT: thermodynamic databases for proteins and protein-nucleic acid interactions.

ProTherm and ProNIT are two thermodynamic databases that contain experimentally determined thermodynamic parameters of protein stability and protein-nucleic acid interactions, respectively. The current versions of both the databases have considerably increased the total number of entries and enhanced search interface with added new fields, improved search, display and sorting options. As on September 2005, ProTherm release 5.

Identification of a key amino acid residue of Streptomyces phospholipase D for thermostability by in vivo DNA shuffling.

To isolate thermostability-related amino acid residues of Streptomyces phospholipase D (PLD), we constructed a chimeral genes library between two highly homologous plds, which exhibited different thermostabilities, by an in vivo DNA shuffling method using Escherichia coli that has a mutation of a single-stranded DNA-binding protein gene. To confirm the location of the recombination site, we carried out the restriction mapping of 68 chimeral pld genes. The recombination sites were widely dispersed over the entire pld sequence.

ProTherm, version 4.0: thermodynamic database for proteins and mutants.

Release 4.0 of ProTherm, thermodynamic database for proteins and mutants, contains approximately 14,500 numerical data (approximately 450% of the first version) of several thermodynamic parameters along with experimental methods and conditions, and structural, functional and literature information. The sequence and structural information of proteins is connected with thermodynamic data through links between entries in Protein Data Bank, Protein Information Resource and SWISS-PROT and the data in ProTherm.

Importance of mutant position in Ramachandran plot for predicting protein stability of surface mutations.

Understanding the mechanisms by which mutations affect protein stability is one of the most important problems in molecular biology. In this work, we analyzed the relationship between changes in protein stability caused by surface mutations and changes in 49 physicochemical, energetic, and conformational properties of amino acid residues. We found that the hydration entropy was the major contributor to the stability of surface mutations in helical segments; other properties responsible for size and volume of molecule also correlated significantly with stability.

Thermodynamic databases for proteins and protein-nucleic acid interactions.

Thermodynamic data regarding proteins and their interactions are important for understanding the mechanisms of protein folding, protein stability, and molecular recognition. Although there are several structural databases available for proteins and their complexes with other molecules, databases for experimental thermodynamic data on protein stability and interactions are rather scarce. Thus, we have developed two electronically accessible thermodynamic databases.

ProTherm, Thermodynamic Database for Proteins and Mutants: developments in version 3.0.

The current release of ProTherm, Thermodynamic Database for Proteins and Mutants, contains more than 10 000 numerical data (300% of the first version) of several thermodynamic parameters, experimental methods and conditions, reversibility of folding, details about the surrounding residues in space for all mutants, structural, functional and literature information. In the current version, we have added information about the source of each protein, identification codes for SWISS-PROT and Protein Information Resource and unique Protein Data Bank (PDB) code for proteins with relevant source. We have also provided additional options to search for data based on PDB code, number of states and reversibility.

Role of hydration of polyhydroxy compounds in biological systems.

The spin-lattice relaxation times, T1, of H2(17)O have been measured for aqueous solutions of 9 polyols as a function of the concentration at 25 degrees C. The dynamic hydration number, nDHN, for polyols were obtained. The values of nDHN increased with an increase in the number of OH groups and depended on the conformation of isomers.

Thermodynamic database for protein-nucleic acid interactions (ProNIT).

Motivation: Protein-nucleic acid interactions are fundamental to the regulation of gene expression. In order to elucidate the molecular mechanism of protein-nucleic acid recognition and analyze the gene regulation network, not only structural data but also quantitative binding data are necessary. Although there are structural databases for proteins and nucleic acids, there exists no database for their experimental binding data.

Importance of surrounding residues for protein stability of partially buried mutations.

For understanding the factors influencing protein stability, we have analyzed the relationship between changes in protein stability caused by partially buried mutations and changes in 48 physico-chemical, energetic and conformational properties of amino acid residues. Multiple regression equations were derived to predict the stability of protein mutants and the efficiency of the method has been verified with both back-check and jack-knife tests. We observed a good agreement between experimental and computed stabilities.

ProTherm, version 2.0: thermodynamic database for proteins and mutants.

ProTherm 2.0 is the second release of the Thermo-dynamic Database for Proteins and Mutants that includes numerical data for several thermodynamic parameters, structural information, experimental methods and conditions, functional and literature information. The present release contains >5500 entries, an approximately 67% increase over the previous version.

Folding energetics of a multidomain protein, flagellin.

Thermodynamic investigations of flagellin from Salmonella typhimurium and its proteolytic fragments were conducted by differential scanning calorimetry (DSC) and circular dichroism (CD) melting measurements. A new method of analysis for a multi-state transition based on our original theoretical treatment of thermodynamic equations has been developed to analyze those data. The analysis of DSC curves confirmed the three thermodynamic domains of flagellin.

Shape and energetics of a cavity in c-Myb probed by natural and non-natural amino acid mutations.

The shape and the energetics of a functional cavity in the R2 subdomain (90-141) of the c-Myb DNA-binding domain were investigated by spectroscopy and thermodynamic analysis. We focused on the valine 103 residue located in front of the cavity. Nine mutants, in which valine 103 was substituted with alanine, 2-aminobutyric acid, norvaline, norleucine, leucine, isoleucine, allo -isoleucine, cyclohexylglycine, and cyclohexylalanine, were chemically synthesized and analyzed.

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 beta-strand segments correlated better than did those in alpha-helical segments, which may result from stronger hydrophobicity of the beta-strands.

Role of structural and sequence information in the prediction of protein stability changes: comparison between buried and partially buried mutations.

Predicting mutation-induced changes in protein stability is one of the greatest challenges in molecular biology. In this work, we analyzed the correlation between stability changes caused by buried and partially buried mutations and changes in 48 physicochemical, energetic and conformational properties. We found that properties reflecting hydrophobicity strongly correlated with stability of buried mutations, and there was a direct relation between the property values and the number of carbon atoms.

Crystallization and preliminary X-ray analysis of wild-type and V103L mutant Myb R2 DNA-binding domain.

The R2 subdomain of the mouse c-Myb DNA-binding domain and its V103L mutant have been crystallized by the vapour-diffusion method using highly concentrated sodium citrate at pH 6.8 as a precipitant. Using ammonium sulfate as precipitant in MES buffer only produced crystals for the mutant R2.

Domain organization of flagellar hook protein from Salmonella typhimurium.

Hook forms a universal joint, which mediates the torque of the flagellar motor to the outer helical filaments. Domain organization of hook protein from Salmonella typhimurium was investigated by exploring thermal denaturation properties of its proteolytic fragments. The most stable part of hook protein involves residues 148 to 355 and consists of two domains, as revealed by deconvolution analysis of the calorimetric melting profiles.

Proton nuclear magnetic resonance spectroscopy of urine for rapid multicomponent analysis of intraoperative cellular metabolites.

We serially measured the proton nuclear magnetic resonance spectra of the urine of intraoperative patients over time to assess their potential use for rapid multicomponent analysis of cellular metabolites. Within a few minutes, the spectra provided signals of many low molecular weight urinary metabolites, including amino acids, ketone bodies, lactate, and glucose. The proton magnetic resonance spectra of the urine of most of the intraoperative patients showed large increases in urinary excretion of alanine, ketone bodies, and lactate and/or glucose, confirming alterations in energy substrate-endocrine relationships during the perioperative period.

Fragment reconstitution of a small protein: folding energetics of the reconstituted immunoglobulin binding domain B1 of streptococcal protein G.

To elucidate early stages in protein folding, we have adopted a fragment reconstitution method for small proteins. This approach is expected to provide nuclei for protein folding and to allow us to investigate folding mechanisms. In previous work [Kobayashi, N.

Mechanism of self-association and filament capping by flagellar HAP2.

HAP2 forms a capping structure, which binds very tightly to the distal end of flagellar filaments and still allows insertion of flagellin subunits below the cap by an unknown mechanism. Terminal regions of HAP2 from Salmonella typhimurium were found to be quickly degraded by various proteases, indicating that HAP2 also possesses disordered terminal regions like other axial proteins of bacterial flagellum. Removal of these portions by trypsin results in a fragment of 40 kDa (HP40), which lacks 42 NH2-terminal and 51 COOH-terminal residues.

ProTherm: Thermodynamic Database for Proteins and Mutants.

The first release of the Thermodynamic Database for Proteins and Mutants (ProTherm) contains more than 3300 data of several thermodynamic parameters for wild type and mutant proteins. Each entry includes numerical data for unfolding Gibbs free energy change, enthalpy change, heat capacity change, transition temperature, activity etc., which are important for understanding the mechanism of protein stability.

Special folding pathway to tetramer only through the micelle state of the corticotropin-releasing factor.

The ovine corticotropin-releasing factor (CRF), a peptide hormone of 41 residues stimulating the secretion of adrenocorticotropic hormone, was thermodynamically investigated. By means of size exclusion chromatography and/or ultrafiltration, the CRF solution could be separated into random coil monomers and highly alpha-helical tetramers, which seem to have amphipathic helix bundle structure. Circular dichroism measurements along with diluting or concentrating the CRF solution revealed that there exists the micelle state above the concentration of 0.

Structural organization and assembly of flagellar hook protein from Salmonella typhimurium.

The terminal regions of monomeric hook protein from Salmonella typhimurium are known to be highly mobile and exposed to the solvent. Although hook protein exhibits an unusual far-UV circular dichroism spectrum, resembling that of random coil structures, our calorimetric experiments clearly demonstrate that the molecule has a compact ordered core. The compact part probably consists of three domains as suggested by deconvolution analysis of the calorimetric melting profiles.

Complement assembly of two fragments of the streptococcal protein G B1 domain in aqueous solution.

We examined the complementation of various pairs of fragments derived from the streptococcal protein G B1 domain by NMR and CD. Most were not associated; however, one pair of fragments (1-40) and (41-56) interacted sufficiently enough to regenerated a stable 1:1 complex, Kd = 9 x 10(-6) M. A 2D-NMR analysis showed that the structure of the complex resembled that of native domain.

Proton NMR spectroscopic studies of serum as an aid to perioperative cellular metabolic monitoring.

Perioperative concentration changes of cellular metabolites in serum were studied by proton NMR spectroscopy in four cancer patients who underwent tumorectomies under general anesthesia. In proton NMR spectra of serum, the resonances assignable to fatty acid in lipoprotein, lactate, alanine, glucose, glycoprotein and other metabolites were observed. The concentrations of fatty acid and alanine did not show significant changes during the operations compared with those in healthy volunteers.