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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.

7,8-Dihydro-8-oxoguanine (8-oxoG) is the most common form of oxidative DNA damage in human cells. Biochemical studies have shown that 8-oxoG decreases the DNA cleavage activity of human topoisomerase I, an enzyme vital to DNA metabolism and stability. We present the 3.1-[Angstrom] crystal structure of human topoisomerase I in noncovalent complex with a DNA oligonucleotide containing 8-oxoG at the +1 position in the scissile strand. We find that 8-oxoG reorganizes the active site of human topoisomerase I into an inactive conformation relative to the structures of topoisomerase I-DNA complexes elucidated previously. The catalytic Tyr-723-Phe rotates away from the DNA cleavage site and packs into the body of the molecule. A second active-site residue, Arg-590, becomes disordered and is not observed in the structure. The docked, inactive conformation of Tyr-723-Phe is reminiscent of the related tyrosine recombinase family of integrases and recombinases, suggesting a common regulatory mechanism. We propose that human topoisomerase I binds to DNA first in an inactive conformation and then rearranges its active site for catalysis. 8-OxoG appears to impact topoisomerase I by stabilizing the inactive, DNA-bound state.

Research examines the solution structure and the process of protein unfolding of bovine adrenodoxin by steady-state kinetics analyses of tryptophan fluorescence. In mutants, in which the phenylalanine or tyrosine of adrenodoxin is replaced with tryptophan, the steady-state and time-resolved unfolding properties correlated with those derived from the crystal structure.

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.

Structural and charge interaction analyses of the binding site of the aspartate 95 mutants of the Desulfovibrio vulgaris flavodoxin reveals the role played by the hydrogen bonding in the modulation of the redox potentials of the bound flavodoxin, particularly the carboxylate group of the mutant.

Analyses of the oligomerization and functional interactions of the B ephrin cytoplasmic domain by nuclear magnetic resonance spectroscopy reveals that the entire domain is incapable of folding into three-dimensional structures and hence prone to aggregation. Further, the two structural elements of the C-terminal functional subdomain exhibit unique backbone motion.

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.

The models for the binding of apolipoprotein-1 to discoidal high-density lipoprotein particles are reviewed using the fluorescence resonance energy transfer along with the proteolysis data. The belt or hairpin models are preferred over the picket fence model for particles of 105 angstrom diameter.

Nuclear magnetic spectroscopic analysis demonstrates that the alpha(sub)IIb and beta(sub)3 cytoplasmic tails interact, in a weak handshake fashion, which restrains the integrin in a resting state. Further, unclasping of this interaction by 'activating' mutations lead to an inside-out conformation and receptor activation.

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.

Site-directed mutagenesis of the actin reveals that the hydrophobic loop comprising the amino acid residues 262-274 plays a crucial role in the polymerization of actin into filaments. Data show that the disulfide cross-linked actin mutant forms amorphous aggregates instead of normal filaments with reduced actin.

Results indicate that amino acid residues R71, D75, and H98 mediate the stabilization of the semiquinone state in the high-affinity binding site in cytochrome bo(sub)3 of Escherichia coli.

Results point out that Escherichia coli leucyl-tRNA synthetase modified by replacement of T252 amino acid residue shows attenuated editing activity toward natural amino acids. However, the mutant enzyme incorporates unsaturated amino acids into proteins facilitating incorporation of unique protein building blocks.

Results point out that all luteovirid P1-P2 pseudoknots assume folded structures maximizing cooperativity and complementarity of L1-S2 and L2-S1 loop-stem interactions involved in the stabilization of the pseudoknotted conformation relative to those of partially folded and inactive stem-loop structures.

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.

Escherichia coli leucyl-tRNA synthetase mutants at amino acid E292 do not differ from wild-type in structure but exhibit impaired enzyme activity. Data suggest that decreased aminoacylation and editing activity of the mutants and tRNA(sup)Leu isoacceptor discrimination of editing in mutants are influenced by E292 mutation.

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.

Research reveals that the energetic state of the intra A-chain/domain disulfide in insulin and insulin-like growth factor 1 is not regulated by the chain/domain sequence but is controlled by the B-chain/domain sequence.

Research reveals that the high affinity and specificity of the human Puf protein, Pumilio1, homology domain is mediated by the multiple copies of a repeat motif of the protein to which RNA binds through three amino acid side chains.

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 covalent attachment of ubiquitin is an important determinant for selective protein degradation by the 26S proteasome in plants and animals. The specificity of ubiquitination is often controlled by ubiquitin-protein ligases (or E3s), which facilitate the transfer of ubiquitin to appropriate targets. One ligase type, the SCF E3s are composed of four proteins, cullin1/Cdc53, Rbx1/Roc1/Hrt1, Skp1, and an F-box protein. The F-box protein, which identifies the targets, binds to the Skp1 component of the complex through a degenerate N-terminal [approximately equal to] 60-aa motif called the F-box. Using published F-boxes as queries, we have identified 694 potential F-box genes in Arabidopsis, making this gene superfamily one of the largest currently known in plants. Most of the encoded proteins contain interaction domains C-terminal to the F-box that presumably participate in substrate recognition. The F-box proteins can be classified via a phylogenetic approach into five major families, which can be further organized into multiple subfamilies. Sequence diversity within the subfamilies suggests that many F-box proteins have distinct functions and/or substrates. Representatives of all of the major families interact in yeast two-hybrid experiments with members of the Arabidopsis Skp family supporting their classification as F-box proteins. For some, a limited preference for Skps was observed, suggesting that a hierarchical organization of SCF complexes exists defined by distinct Skp/F-box protein pairs. Collectively, the data shows that Arabidopsis has exploited the SCF complex and the ubiquitin/26S proteasome pathway as a major route for cellular regulation and that a diverse array of SCF targets is likely present in plants.

[Na.sup.+]/[H.sup.+] exchanger regulatory factor (NHERF)-1 and NHERF-2, two structurally related protein adapters containing tandem PSD-95/ Discs large/ZO-1 (PDZ) domains, were identified as essential factors for protein kinase A-mediated inhibition of the sodium-hydrogen exchanger, NHE3. NHERF-1 and NHERF-2 also bound other cellular targets including the sodium-phosphate cotransporter type IIa encoded by the NPT2 gene. Targeted disruption of the mouse NHERF-1 gene eliminated NHERF-1 expression in kidney and other tissues of the mutant mice without altering NHERF-2 levels in these tissues. NHERF-1 (+/-) and (-/-) male mice maintained normal blood electrolytes but showed increased urinary excretion of phosphate when compared with wild-type (+/+) animals. Although the overall levels of renal NHERF-1 targets, NHE3 and Npt2, were unchanged in the mutant mice, immunocytochemistry showed that the Npt2 protein was aberrantly localized at internal sites in the renal proximal tubule cells. The mislocalization of Npt2 paralleled a reduction in the transporter protein in renal brush-border membranes isolated from the mutant mice. In contrast, NHE3 was appropriately localized at the apical surface of proximal tubules in both wild-type and mutant mice. These data suggested that NHERF-1 played a unique role in the apical targeting and/or trafficking of Npt2 in the mammalian kidney, a function not shared by NHERF-2 or other renal PDZ proteins. Phosphate wasting seen in the NHERF-1(-/-) null mice provided a new experimental system for defining the role of PDZ adapters in the hormonal control of ion transport and renal disease.

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.

Benzophenones are among the most useful photocrosslinking agents in biology. We have evolved an orthogonal aminoacyl-tRNA synthetase/tRNA pair that makes possible the in vivo incorporation of p-benzoyl-L-phenylalanine into proteins in Escherichia coli in response to the amber codon, TAG. This unnatural amino acid was incorporated with high translational efficiency and fidelity into the dimeric protein glutathione S-transferase. Irradiation resulted in efficient crosslinking (>50%) of the protein subunits. This methodology may prove useful for discovering and defining protein interactions in vitro and in vivo.

The 18:0-holo-acyl carrier protein delta(sup)9 desaturase reactivity is probed using fatty acids with single oxygen atom replacing the methylene groups in a C-18 fatty acyl chain. Results demonstrate desaturation, acyloxy chain scission, and 'register-shift' in binding of substrate. Data indicate that the desaturase-catalyzed reactions may initiate at the C-10 position.

the accessibility of the FLAG epitope tag, introduced into the three membrane-spanning domains of the human multidrug resistance protein transporter, MRP1, depends on the location of the tag and the glycosylation sites in the membrane-spanning domains 1 and 3. The cell surface glycosylation may play a role in masking the extracellular epitopes of the transporter.

The antimicrobial cyclic peptide, RTD-1, exhibits two physically distinct states of binding to lipid bilayers as shown by circular dichroism and X-ray lamellar diffraction techniques. However, RTD-1 shows differences in the nature of transition between the two states in contrast with other antimicrobial peptides. RTD-1 induced membrane thinning depends on the peptide-to-lipid-ratio.

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 ultraviolet resonance Raman analysis of the nickel-iron hybrid hemoglobin reveals differences in structural changes at the alpha1-beta2 subunit interface between ligand binding to the alpha and beta hemes and influenced by inositol-hexa(kis)phosphate.

The fluorescence analysis reveals an activation-dependent conformational change in the G protein alpha leading to increased hydrophobic environment of the N-terminal region. The conformational changes occur upon G protein betagamma binding and activated to either the GDP-AlF(sub)4(sup)- or the GTP-gammaS bound conformation.

Results reveal that the incorporation of the tumor-associated Tn carbohydrates at Thr3 and Ser4 upstream from the MUCI muscin core peptide epitope of the sequence PDTRP increases antibody B27.29 binding affinity through carbohydrate-antibody interaction. MUC1 mucin is a transmembrane glycoprotein with a 20 amino acid repeat sequence.

Results point out that the AT-rich DNA sequences, containing divalent manganese in the minor groove, can be modulated by base pair changes affecting both the width and electrostatic potential of the mionor groove.

Research illustrates the aggregation of barstar protein at low pH proceeding via the soluble aggregate A form. Data show that the aggregation process continues for several weeks, finally terminating in an insoluble aggregate. The N-terminal segment of the A form aggregated core exhibits free-flight motion.

Research suggests that the urea equilibrium unfolding of the hepatitis B surface antigen single-chain Fc antibody fragment is four-state, which is also the case during thermal unfolding but with different intermediary states. Global analyses of both thermal- and urea-mediated unfolding are presented.

The nuclear magnetic resonance analysis suggests a toroidal model for the mechanism of action of protegrin-1 and the model is consistent with neutron and X-ray diffreaction and circular dichroism of protegrin-1 with magainin. Results also reveal a dramatic alteration in the lipid orientation and headgroup conformation at protein:lipid ratio greater than 1:30.

Results reveal that there exists a functional metal ion selectivity in the Synechococcus PCC7942 SmtB family of metal sensor proteins. Cysteine substitution or derivatization in the alpha3N metal binding site does not affect allosteric negative regulation by divalent zinc, while zinc-ligand substitution in the alpha5 metal binding site renders SmtB refractory to SmtB-DNA complexes.

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.

Research reveals that the branched-chain amino acid permease from Rhizobium leguminosarum transports polar amino acids, gamma-aminobutyric acid, hydrophobic-, and neutral amino acids, suggesting that it functions as a global amino acid transporter.

Results point out that chromatin architecture that is inhibitory to transcription is a controlling factor of gene expression and overrides the influence of receptor-ligand, protein-protein, and protein-DNA interactions on the gene expression. The activators bind to their sites in the genome where the chromatin is unaltered and initiate transcription as exemplified by interferon-beta gene.

Early conformational events during folding of acyl-CoA binding protein (ACBP), an 86-residue [alpha]-helical protein, were explored by using a continuous-flow mixing apparatus with a dead time of 70 [micro]s to measure changes in intrinsic tryptophan fluorescence and tryptophan-dansyl fluorescence energy transfer. Although the folding of ACBP was initially described as a concerted two-state process, the tryptophan fluorescence measurements revealed a previously unresolved phase with a time constant [tau] = 80 [micro]s, indicating formation of an intermediate with only slightly enhanced florescence of Trp-55 and Trp-58 relative to the unfolded state. To amplify this phase, a dansyl fluorophore was introduced at the C terminus by labeling an 186C mutant of ACBP with 5-IAEDANS [5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid]. Continuous-flow refolding of guanidine HCl-denatured ACBP showed a major increase in tryptophan-dansyl fluorescence energy transfer, indicating formation of a partially collapsed ensemble of states on the 100-[micro]s time scale. A subsequent decrease in dansyl fluorescence is attributed to intramolecular quenching of donor fluorescence on formation of the native state. The kinetic data are fully accounted for by three-state mechanisms with either on- or off-pathway intermediates. The intermediate accumulates to a maximum population of 40%, and its stability depends only weakly on denaturant concentration, which is consistent with a marginally stable ensemble of partially collapsed states with ~1/3 of the solvent-accessible surface buried. The findings indicate that ultrafast mixing methods combined with sensitive conformational probes can reveal transient accumulation of intermediate states in proteins with apparent two-state folding mechanisms.

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

Results show that the binding of 6-hydroxymethyl-7,8-dihydropterin by 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase is medited by a step or steps after the chemical transformation and serve as rate-limiting steps of the reactions catalyzed by the enzyme and magnesium.

Results reveal that a Trp residue in the N-terminus of epidermal growth factor-like growth factors enhances the binding affinity for ErbB-3 receptor. Furthermore, epidermal growth factor variants, NRG-1 and TGF-alpha, exhibit high binding affinity for ErbB-2/ErbB-3 heterodimers as well as for ErbB-3 alone.

Electron paramagnetic resonance spectroscopic analysis of the steady-state radical intermediate in the deamination of the amino alcohol S-2-aminopropanol reveals that the electron spin-spin coupling between the radical and the low-spin Co(sup)2+ in cobalt(II)alamin (B(sub)12r) are separated by approximately 11 angstrom.

Results reveal that some polymers display pi-aggregates resembling edge-on-structure at the air-water interface, while monolayer Langmuir-Blodgett films of the polymers show a reduction in the quantum yields. Polymers that do not form pi-aggregates exhibit a highly emissive face-on-structure at the air-water interface.

The development of three-dimensional maps of the modified nucleotides in the ribosomes of Escherichia coli and yeast has revealed that most (~95% in E. coli and 60% in yeast) occur in functionally important regions. These include the peptidyl transferase centre, the A, P and E sites of tRNA- and mRNA binding, the polypeptide exit tunnel, and sites of subunit-subunit interaction. The correlations suggest that many ribosome functions benefit from nucleotide modification.

A novel domain (the BRICHOS domain) of ~100 amino acids has been identified in several previously unrelated proteins that are linked to major diseases. These include BR[I.sub.2], which is related to familial British and Danish dementia (FBD and FDD); Chondromodulin-I (ChM-I), related to chondrosarcoma; CA11, related to stomach cancer; and surfactant protein C (SP-C), related to respiratory distress syndrome (RDS). In several of these, the conserved BRICHOS domain is located in the propeptide region that is removed after proteolytic processing. Experimental data suggest that the role of this domain could be related to the complex post-translational processing of these proteins.

Research describes the synthesis, isolation, and structural characterization and magnetic/electron paramagnetic resonance analyses of a complex between cobalt(II) and the diphosphonate ligand, imino-bis(methylphosphonic acid). Data indicate that the complex possesses solid-state properties of the metal organophosphonate material and mononuclear Co(II) sites.

Research demonstrates the antimicrobial activity of the 12-helical beta-peptides. Data indicate a 40% cationic face is optimum for their activity and those with higher cationic face are inactive. The mechanism of action of beta-peptides is similar to those of antimicrobial alpha-peptides and they are impervious to proteases.