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Results point out that the Neurospora SET domain protein DIM-5 does not exhibit any structural similarity to the AdoMet-dependent methyltransferases. However, it possesses zinc cluster in the pre-SET domain and at the AdoMet binding site in the SET domain, which are involved in methyl transfer.

A novel FAD-binding domain, BLUF, exemplified by the N-terminus of the AppA protein from Rhodobacter sphaeroides, is present in various proteins, primarily from Bacteria. The BLUF domain is involved in sensing blue-light (and possibly redox) using FAD and is similar to the flavin-binding PAS domains and cryptochromes. The predicted secondary structure reveals that the BLUF domain is a novel FAD-binding fold.

The conformational changes in Ras that accompany the hydrolysis of GTP are critical to its function as a molecular switch in signaling pathways. Understanding how GTP is hydrolyzed by revealing the sequence of intermediary structures in the reaction is essential for understanding Ras signaling. Until now, no structure of an intermediate in GTP hydrolysis has been experimentally determined for Ras alone. We have solved the crystal structure of the Ala-59 to Gly mutant of Ras, (RasA59G), bound to guanosine 5'-imidotriphosphate or GDP to 1.7-[Angstrom] resolution. In the guanosine 5'-imidotriphosphate-bound form, this mutant adopts a conformation that is intermediate between the GTP- and GDP-bound forms of wild-type Ras and that is similar to what has been predicted by molecular dynamics simulation [Ma, J. P. & Karplus, M. (1997) Proc. Natl. Acad. Sci. USA 94, 11905-11910]. This conformation is stabilized by direct and water-mediated interactions between the switch 1 and switch 2 regions and is characterized by an increase in the binding affinity for GTP. We propose that the structural changes promoted by the Ala-59 to Gly mutation exhibit a discrete conformational state assumed by wild-type Ras during GTP hydrolysis.

Results describe the structure of the complex between memapsin 2 catalytic domain and the inhibitor OM99-2 and identify two new subsites and binding modes of the P(sub)2 and P(sub)4 side chains. The new information is useful in the design of effective inhibitors.

The two-component receiver domain of Sinorhizobium meliloti sigma(sup)54-dependent AAA+ ATPase, DctD, binds BeF(sub)3(sup)- at the aspartate carboxylic group and mimics the protein's phosphorylation leading to signal transduction by DctD. The crystal lattice for magnesium-BeF(sub)3(sup)- --DctD fragment exhibits multiple 'dimers'. Binding of the magnesium-BeF(sub)3(sup)- leads to marked changes in the dimeric state of DctD.

Results reveal that of the two sites, S(I-299) or E(II-101), involved in the entry point of the potassium-pathway, only the latter is associated with the entry point pathway. Mutation of E(II-101) leads to impaired proton release but does not affect the P(sub)R formation kinetics.

Three-dimensional solution structure of ascidian trypsin inhibitor exhibits an alpha-helical structure formed by the Cys-X1-X2-X3-Cys segment representing amino acid residues 37-41. Data indicate that the secondary structure and main chain overall folding is similar to those of Kazal-type inhibitors.

Substitution of proline residues with alanine in the P3'-P4' cis-Pro-trans-Pro sequence of the Bowman-Birk inhibitor family of protease inhibitors reveals that the proline at P3' and a cis peptide bond at that position are essential for the biological activity. Further, P4' stabilizes the cis conformation.

Numerous bacterial pathogens use type IV secretion systems (T4SS) to deliver virulence factors directly to the cytoplasm of plant, animal, and human host cells. Here, evidence for interactions among components of the Agrobacterium tumefaciens vir-encoded T4SS is presented. The results derive from a high-resolution yeast two-hybrid assay, in which a library of small peptide domains of T4SS components was screened for interactions. The use of small peptides overcomes problems associated with assaying for interactions involving membrane-associated proteins. We established interactions between VirB11 (an inner membrane pore-forming protein), VirB9 (a periplasmic protein), and VirB7 (an outer membrane-associated lipoprotein and putative pilus component). We provide evidence for an interaction pathway, among conserved members of a T4SS, spanning the A. tumefaciens envelope and including a potential pore protein. In addition, we have determined interactions between VirB1 (a lytic transglycosylase likely involved in the local remodeling of the peptidoglycan) and primarily VirB8, but also VirB4, VirB10, and VirB11 (proteins likely to assemble the core structure of the T4SS). VirB4 interacts with VirB8, VirB10, and VirB11, also establishing a connection to the core components. The identification of these interactions suggests a model for assembly of the T4SS.

The structure of the reaction center from Rhodobacter sphaeroides has been solved by using x-ray diffraction at a 2.55-[Angstrom] resolution limit. Three lipid molecules that lie on the surface of the protein are resolved in the electron density maps. In addition to a cardiolipin that has previously been reported [McAuley, K. E., Fyfe, P. K., Ridge, J. P., Isaacs, N. W., Cogdell, R. J. & Jones, M. R. (1999) Proc. Natl. Acad. Sci. USA 96, 14706-14711], two other major lipids of the cell membrane are found, a phosphatidylcholine and a glucosylgalactosyl diacylglycerol. The presence of these three lipids has been confirmed by laser mass spectroscopy. The lipids are located in the hydrophobic region of the protein surface and interact predominately with hydrophobic amino acids, in particular aromatic residues. Although the cardiolipin is over 15 [Angstrom] from the cofactors, the other two lipids are in close contact with the cofactors and may contribute to the difference in energetics for the two branches of cofactors that is primarily responsible for the asymmetry of electron transfer. The glycolipid is 3.5 [Angstrom] from the active bacteriochlorophyll monomer and shields this cofactor from the solvent in contrast to a much greater exposed surface evident for the inactive bacteriochlorophyll monomer. The phosphate atom of phosphatidylcholine is 6.5 [Angstrom] from the inactive bacteriopheophytin, and the associated electrostatic interactions may contribute to electron transfer rates involving this cofactor. Overall, the lipids span a distance of [approximately equal to] 30 [Angstrom], which is consistent with a bilayer-like arrangement suggesting the presence of an 'inner shell' of lipids around membrane proteins that is critical for membrane function.

Deletion mutants exhibiting truncation of the manganese stabilizing protein by 18 amino acids of the N-terminus renders the protein incapable of binding to photosystem II as well as its reconstitution activity. The N-terminal domain (sup)15T--(sup)18T is required for the binding activity, suggesting unlike eukaryotes, cyanobacteria lack only one binding sequence.

The homodimer of the cysteine substituted mutant SmtB metalloregulatory protein of the Synechococcus PCC7942 binds divalent zinc or cobalt in one of the two metal binding sites, alpha3N and alpha5. Data suggest that the differential binding behavior of the metal binding sites accounts for the functional diversity of this family of proteins.

The mexR gene encoded product MexR in nalB mutants of Pseudomonas aeruginosa is modified by a single amino acid that compromizes the stability of the protein or its ability to dimerize. However, modification of L95 and R21 amino acid residues confer stability and dimerization capability but compromises DNA binding.

Tetrameric hemoglobins are the most widely used systems in studying protein cooperativity. Allosteric effects in hemoglobins arise from the switch between a relaxed (R) state and a tense (T) state occurring upon oxygen release. Here we report the 2.0-[Angstrom] crystal structure of the main hemoglobin component of the Antarctic fish Trematomus newnesi, in a partial hemichrome form. The two [alpha]-subunit iron atoms are bound to a CO molecule, whereas in the [beta] subunits the distal histidine residue is the sixth ligand of the heme iron. This structure, a tetrameric hemoglobin in the hemichrome state, demonstrates that the iron coordination by the distal histidine, usually associated with denaturing states, may be tolerated in a native-like hemoglobin structure. In addition, several features of the tertiary and quaternary organization of this structure are intermediate between the R and T states and agree well with the R [right arrow] T transition state properties obtained by spectroscopic and kinetic techniques. The analysis of this structure provides a detailed pathway of heme-heme communication and it indicates that the plasticity of the [beta] heme pocket plays a role in the R [right arrow] T transition of tetrameric hemoglobins. protein denaturation | protein function | x-ray structure | Antarctic

Research examines the role of Glu194 and Glu204 amino acid residues on the photocycle reactions of bacteriorhodopsin, which mediate proton release from bacteriorhodopsin to the exterior. Low-temperature FTIR difference spectra of the mutant proteins, as influenced by chloride at different pH conditions, reveal that mutations or chloride affect the second part of the photocycle.

Results show a stepwise enhancements in alpha-helicity upon sodium and D-galactose binding by the prokaryotic sodium/galactose transporter from Vibrio parahaemolyticus. Data reveal that helicity increases are accompanied by decreases in beta-structural elements and degree of H/D exchange, indicating reduced water accessibility to the protein.

The on-line pseudo cell-size exclusion chromatography--mass spectrometry analysis shows that the transition-state regulator AbrB is assembled as a homotetramer and binds to the sin/R DNA target element in a 4:1 protein:DNA stoichiometry. Data indicate that when AbrB complex binds to sinIR, there is a reduction in the hydrodynamic volume, suggesting a conformational change.

The designed mini agouti-related protein, containing a 34-residue and a cystine knot architecture, functions as an antagonist with the melanocortin receptor contact points. This new molecule is useful for probing structure and function of melanocortin receptor.

The structure of the the primary constituent of paired helical filaments, the tau-protein, exhibits alpha-helices containing an abundance of helix-breaking amino acids with no solution structure. Further, the paired helical filaments are stable, resistant to protease and their aggregation forms alpha-helix.

Research shows that the small molecule N-(benzene-sulfonyl)-(L)-prolyl-(L)-O-(1-pyrrolidinylcarbonyl)tyrosine is a potent inhibitor of alpha9beta1 and alpha4beta1 integrins. Data reveal differences in the structure and function of the integrins but interestingly they exhibit very similar ligand binding and metal ion dependencies for their activation.

The X-ray crystal analysis of human c8gamma reveals a typical lipocalin fold forming a calyx and exhibiting a binding site for a small molecule, whose identity is unknown. C8gamma is a subunit of human C8 which in turn a component of the cytolytic membrane attack complex of complement.

The calcium-binding protein, centrin, exhibits conformational changes upon calcium binding. The amide I' band and the side-chain aspartate absorbance are localized to the calcium-binding sites. Data indicate that the secondary structure of centrin influences the amide I' band in the presence and absence of calcium.

Results reveal that the Escherichia coli multidrug transporter MdfA retains its activity of drug resistance and transport in the absence of the entire C-terminal transmembrane domain and the C terrminus as indicated by complementary gene fusion between truncated MdfA-Cat and MdfA-PhoA. The MdfA belongs to the 12-transmembrane-domain family of transporters.

Structural analysis of an 81-amino acid cytochrome c from Shewanella puttrefaciens reveals a structure with rmsd values from the mean 0.50 and 0.96 angstrom for backbone and heavy atoms, respectively. Thirty percent of the sequences, in out of 10 different cytochromes c sequences, show identity to the bacterial cytochrome.

A working model for iron-sulfur cluster assembly protein is presented using the recombinat purified yeast protein. Data suggest that yeast protein is sparingly soluble at ambient temperatures, is sensitive to proton-mediated degradation, and forms a complex with the electron transport protein ferredoxin in both apo and holo states.

The crystal structure of the diheme cytochrome NapB from Haemophilus influenzae shows an unusual fold with two heme groups exhibiting nearly parallel heme planes and iron-to iron distance of only 9.9 angstroms. Data indicate that the structural organization of the heme groups resemble that of split-Soret diheme cytochrome c but folded differently.

The transient grating signals of the histidine mutants of myoglobin are sensitive to the amino acid residue position 64, which is due to ligand dissociation from the protein. The role of histidine 64 of carboxymyoglobin in the photodissociation process, in terms of volume and enthalpy changes, is discussed.

The three-dimensional structure of L-threonine-O-3-phosphate decarboxylate complexed with the pyridoxal 5'-phosphate reveals that the structure is very similar to that of aspartate- and histidinol aminotransferases, suggesting an evolutionary trend within the conserved class of aminotransferases.

The high-resolution structure of the MtH895 protein from the thermophilic archeon Methanobacterium thermoautotrophicum reveals that it is the smallest known member of the thioredoxin family of proteins. The solution structure, by NMR spectroscopy, of MtH895 shows that MtH895 has about 34-44% sequence identitiy with other archaebacteria proteins.

Dendritic microenvironments are analogous to local environments created within protein superstructures. Correspondingly, properties of functional cores such as molecular recognition and catalytic activity are profoundly influenced by the surrounding dendritic branches.

Selenoprotein R (SelR) is a mammalian selenocysteine-containing protein with no known function. Here we report that cysteine homologs of SelR are present in all organisms except certain parasites and hyperthermophiles, and this pattern of occurrence closely matches that of only one protein, peptide methionine sulfoxide reductase (MsrA). Moreover, in several genomes, SelR and MsrA genes are fused or clustered, and their expression patterns suggest a role of both proteins in protection against oxidative stress. Consistent with these computational screens, growth of Saccharomyces cerevisiae SelR and MsrA mutant strains was inhibited, and the strain lacking both genes could not grow, in the presence of [H.sub.2][O.sub.2] and methionine sulfoxide. We found that the cysteine mutant of mouse SelR, as well as the Drosophila SelR homolog, contained zinc and reduced methionine-R-sulfoxide, but not methionine-S-sulfoxide, in in vitro assays, a function that is both distinct and complementary to the stereo-specific activity of MsrA. These findings identify a function of the conserved SelR enzyme family, define a pathway of methionine sulfoxide reduction, reveal a case of convergent evolution of similar function in structurally distinct enzymes, and suggest a previously uncharacterized redox regulatory role of selenium in mammals.

The A[alpha] mating-type locus of Schizophyllum commune regulates sexual development and contains the code for two proteins, Y and Z, which are thought to form a complex and function as a transcription factor. Import of these proteins into the nucleus may be an essential step in A[alpha]-regulated sexual development. The Y proteins contain a bipartite basic sequence, which is an excellent candidate for a nuclear localization sequence (NLS), while Z proteins contain no such sequence. Here we describe experiments in which deletions were made in the putative NLS sequence of Y4. We show that this putative NLS is essential to the function of the Y protein and capable of mislocalizing green fluorescent protein (GFP) to the nucleus in Saccharomyces cerevisiae. Further, we describe genetic experiments that demonstrate the first Y-Y protein interactions in vivo. These results are consistent with our previously postulated hypothesis that the Y-Z complex is likely to be of a higher order than dimer.

Results demonstrate that C-terminal truncations of SecY protein translocase, spanning six to seven residues, affect the insertion of the preprotein signal sequence into the membrane. Data indicate that the truncations do not affect SecA binding but impairs the SecA-dependent translocation initiation.

Single and double substitution mutations in the N-terminus of helix I of the human S100B EF-hand calcium-binding protein reveal that the mutants are less stable than the wild-type with similar trend in the protein unfolding. However the calcium-bound mutants are markedly stabler than calcium-saturated S100B.

Research reveals that removal of 35 residues from the N-terminus of the rusticyanin renders the protein insoluble above pH 5 and the precipitate in solution exhibits an oligomeric form. Data show that the beta-sructure is retained and exposed residues of the deletion mutant are less acidic.

Nuclear magnetic resonance analyses of wild-type alpha2A adrenergic receptor intracellular loop and a mutant peptide, in a micellar environment, show upto 12 residues in the transmembrane 3 and 4 helices flanking the loop. Further, a novel cytoplasmic helix in the alpha2A loop is detected.

Results demonstrate that the second extracellular loop 2 of rhodopsin renders the chromophore-binding domain inaccessible to solvent. The glutamine 181 makedly affects the retinylidene Schiff base of the active metarhodopsin II.

Likelihood estimates of local rates of evolution within proteins reveal that selective constraints on structure and function are quantitatively stable over billions of years of divergence. The stability of constraints produces an intramolecular clock that gives each protein a characteristic pattern of evolutionary rates along its sequence. This pattern allows the identification of constrained regions and, because the rate of evolution is a quantitative measure of the strength of the constraint, of their functional importance. We show that results from such analyses, which require only sequence alignments, are consistent with experimental and mutational data. The methodology has significant predictive power and may be used to guide structure-function studies for any protein represented by a modest number of homologs in sequence databases.

A method for optimizing potential-energy functions of proteins is proposed. The method assumes a hierarchical structure of the energy landscape, which means that the energy decreases as the number of native-like elements in a structure increases, being lowest for structures from the native family and highest for structures with no native-like element. A level of the hierarchy is defined as a family of structures with the same number of native-like elements (or degree of native likeness). Optimization of a potential-energy function is aimed at achieving such a hierarchical structure of the energy landscape by forcing appropriate free-energy gaps between hierarchy levels to place their energies in ascending order. This procedure is different from methods developed thus far, in which the energy gap and/or the Z score between the native structure and all non-native structures are maximized, regardless of the degree of native likeness of the non-native structures. The advantage of this approach lies in reducing the number of structures with decreasing energy, which should ensure the searchability of the potential. The method was tested on two proteins, PDB ID codes 1FSD and 1IGD, with an off-lattice united-residue force field. For 1FSD, the search of the conformational space with the use of the conformational space annealing method and the newly optimized potential-energy function found the native structure very quickly, as opposed to the potential-energy functions obtained by former optimization methods. After even incomplete optimization, the force field obtained by using 1IGD located the native-like structures of two peptides, 1FSD and betanova (a designed three-stranded [beta]-sheet peptide), as the lowest-energy conformations, whereas for the 46-residue N-terminal fragment of staphylococcal protein A, the native-like conformation was the second-lowest-energy conformation and had an energy 2 kcal/mol above that of the lowest-energy structure.

Transmembrane peptides from the receptor tyrosine kinase, ErbB-2, at high concentrations exhibit prominent spectra of rapidly rotating transmembrane monomers and slowly rotating large oligomers. At low concentration of the peptides, the oligomer spectra are diminished but a population of homodimers and/or small oligomers appears, suggesting the peptides possess interaction sites.

This review focuses on the recent methodologies employed to analyze the conformational and structural dynamics of oligosaccharides in glycocproteins. Results indicate that a concerted approach using NMR spectroscopy, X-ray cystallography, and molecular modeling is complementary and reveal a clearer picture of oligosaccharides.

This article summarizes recent advances in the methodology for glycoprotein analysis and structural elucidation of their glycan constituents. The separation methodology is coupled to mass spectrometry and powerful nuclear magnetic resonance spectrometry to increase the sensitivity for structural analysis.

This article discusses the architectural, topofunctional, and molecular aspects of N-linked oligosaccharide biosynthesis in the endoplasm reticulum and the golgi apparatus. The quality aspects of the glycosylated proteins along with the role of golgi apparatus and oligosaccharide structures to cell surface glycoproteins are reviewed.

The cyclin-dependent kinase subunit family of cell cycle regulatory human proteins, ckshs1 and ckshs2, exist as a monomer and a domain-swapped dimer and the kinetic barrier separating these two native states is due to the requirement of protein unfolding for beta-strand exchange to occur.

This article describes the X-ray crystal structure of a serine oxygen ligated [4Fe-4S] cluster of Chromatium vinosum C77S high potential iron protein (HiPIP) at 0.11 angstrom resolution. Data indicate that phenylalanine 48 controls solvent access to the cluster and serine ligation decreases cluster stability.

The solution structure of the calcium-binding protein apo-S100A1 shows that it is a homodimeric protein held together by an X-type four-helix bundle. Data indicate that upon calcium binding, helix 3 changes conformation facilitating S100A1 interaction with other dimeric proteins.

Results point out that disruption of the preorganized alpha-helix of the p27(sup)Kip1 inhibition domain in the unbound state does not alter inhibitory action. However, stabilizing the preformed helix by alanine substitution decreases the rate of inhibition of cyclin-dependent kinase activity by P27.

The interplay between structure-search of the native structure and desolvation in protein folding has been explored using a minimalist model. These results support a folding mechanism where most of the structural formation of the protein is achieved before water is expelled from the hydrophobic core. This view integrates water expulsion effects into the funnel energy landscape theory of protein folding. Comparisons to experimental results are shown for the SH3 protein. After the folding transition, a near-native intermediate with partially solvated hydrophobic core is found. This transition is followed by a final step that cooperatively squeezes out water molecules from the partially hydrated protein core.

The secondary structure of human apolipoprotein(a) KIVT6 consists of antiparallel beta-sheet , a parallel beta-sheet, and a helix containing residues Trp62-Tyr64 and Trp72-Tyr74, Cys1-Tyr2 and Thr78-Gln79, and Thr38-Tyr40, respectively.

Results show that the tubulin-like loop is part of the active site of the prokaryotic cell division protein FitZ, a tubulin homologue, and is involved in GTP hydrolysis mediated by FitZ. Data indicate that FitZ is formed by two of its own monomers.