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The histidine kinase, CheA, couples environmental stimuli to changes in bacterial swimming behavior, converting a sensory signal to a chemical signal in the cytosol via autophosphorylation. The kinase activity is regulated in the platform of chemotaxis signaling complexes formed by CheW, chemoreceptors, and the regulatory domain of CheA. Our previous computational and mutational studies have revealed that two interdomain linkers play important roles in CheA's enzymatic activity. Of the two linkers, one that connects the dimerization and ATP binding domains is essential for both basal autophosphorylation and activation of the kinase. However, the mechanistic role of this linker remains unclear, given that it is far from the autophosphorylation reaction center (the ATP binding site). Here we investigate how this interdomain linker is coupled to CheA's enzymatic activity. Using modern nuclear magnetic resonance (NMR) techniques, we find that by interacting with the catalytic domain, the interdomain linker initiates long-range structural and dynamic changes directed toward the catalytic center of the autophosphorylation reaction. Subsequent biochemical assays define the functional relevance of these NMR-based observations. These findings extend our understanding of the chemotaxis signal transduction pathway.

The experimental data on N-terminal Src homology 3 domain of the Drosophila protein (drk SH3) domain folding states is complemented by studying the folding and unfolding kinetics of the N-terminal SH3 domain of the Drosophila protein drk. Since folding pathways of SH3 domain are conserved within the family, computational studies suggest a common folding mechanism for SH3 domains.

NMR spin relaxation techniques are employed to probe the folding-unfolding kinetics of two SH3 domains as a function of pressure so that the changes in partial molar volumes along the folding pathways can be measured. The volumetric data that is obtained are consistent with a similar folding mechanism for both SH3 domains, involving early chain compaction to states that are at least partially hydrated.

A deuterium (super 2)H and carbon (super 13)C NMR relaxation of methyl side chain dynamics in the 82 kDa enzyme malate synthase G (MSG) that is a promising target for the development of new antibiotic agents is presented. The result establishes that detailed, quantitative information about methyl side chain dynamics can be obtained by NMR on proteins with molecular masses on the order of 100 kDa and opens up the possibilities for studies of motion in a large number of important systems.

Nuclear magnetic resonance relaxation dispersion experiments present an avenue to accessing intermediate states along the reaction pathway, providing kinetic, thermodynamic, and structural information for intermediates with small (greater than or equal to ~ 1%) populations at equilibrium. These techniques are employed to study the three-state folding reaction of the G48M Fyn SH3 domain.

A study is conducted to investigate the motional properties of backbone amide and side chain methyl groups in a peptide derived from the phosphorylated Tyr-1021 (pTyr-1021) site of beta-platelet-derived growth factor receptor (PDGFR), both free and in complex with the PLCC SH2 domain, using NMR relaxation experiments. Deuterium spin relaxation experiments establish that the protein-peptide interface is dynamic, and this mobility may play an important in modulating the affinity of the interaction.

Research has been conducted on the calmodulin-regulated serine/threonine kinase. Molecular recognition characterization of calmodulin-regulated serine/threonine carried out with different calmodulin-regulated serine/threonine kinases via the use of dipolar coupling-based NMR approach is described.

Research has been conducted on the T4 lysozyme mutant. The structure, dynamics and energetics of this mutant have been investigated.

15N nuclear magnetic resonance spectroscopy was done to characterize the backbone dynamics of the folded and denatured states of the N-terminal SH3 domain from the adapter protein drk. The results showed little variation in backbone dynamics across the molecules of the folded protein while that of the denatured form exhibited a more extensive and a more heterogeneous dynamics. It was also found that the level of high-frequency motions in the folded molecule is largely unaffected by variations in temperature. The high-frequency motion of the unfolded protein, on the other hand, is positively correlated with temperature.

Experimental and simulated distance and dihedral restraint sets were used to illustrate the quality of structures that are available from NMR analyses of deuterated, methyl-protonated proteins. As a result of the lower numbers of interproton distance restraints available from these systems, the precision of these structures is substantially lower than what can be obtained from studies of smaller 15N, 13C, 1H-labeled proteins. However, the quality of structures produced is useful for a applications such as topology recognition and guiding site-specific mutagenesis experiments.

Motile bacteria such as Escherichia coli possess the ability to change their swimming behavior in response to environmental changes. This ability is made possible by the chemotaxis signal transduction pathway of E. coli. In this pathway, the histidine autokinase CheA plays a vital role as it aids in the transmission of signals. The structure of this chemotaxis kinase is determined using magnetic nuclear resonance spectroscopy.

The major coat protein of the filamentous bacteriophage IKe in fully protonated myristoyllysophophatidylglycerol (MPG) micelles was analyzed for its structure and chemical dynamics. The IKe coat protein is mostly alpha-helical, with a long amphiphatic surface helix and a short 'micelle-spanning' C-terminal helix. The Pro 30 residue at the C-terminal helix confers hydrogen bonding and stearic restrictions to the molecule.

The dynamics of interactions between a phosphoinositol-specific phospholipase (PLCC) and a pTyr-containing peptide were studied using a nuclear magnetic resonance spin-relaxation technique. The goal was to compare the behavior of a high-affinity, high-specificity binding site with a low-affinity, low specificity binding site in a protein-protein interface. The results show that the high-affinity, high-specificity binding site of PLCC has a very high degree of mobility in the free and peptide-complexed form compared to the low-affinity, low-specificity binding site.

Nuclear magnetic resonance of a 14kDa fragment of CheA, the histidine autokinase in Escherichia (E.) coli that binds the response regulator CheY, reveals that the region has four antiparallel beta-strands. The residues 124 to 257 that make up the CheY region have a structured portion of 68 residues, from 160 to 227, and two unstructured regions at both the ends, referred as domain linkers. CheA is necessary for chemotaxis signal transduction in E. coli.

A method to study the interaction between the phosphopeptide (pY1021) and the arginine residues of the C-terminal of the Src homology 2 domain of phospholipase C-gamma-1 using (super 15)N and (super 13)C NMR relaxation data of the N(super epsilon) and C(super zeta) spins is given. The results of the study show that some arginine residues interact with the phosphorylated tyrosine (pTyr) while others do not interact. Protons are donated by both the NH2 groups of Arg 37. The guanidino groups of Arg 39, 59 and 18 interact with the pTyr ring.

A study was conducted to determine the solution structure of a 110 amino acid cellulose-binding domain from Cex, a beta-1,4-glycanase from the bacterium Cellulomonas fimi. Multidimensional, multinuclear nuclear magnetic resonance spectroscopy was used in tandem with dynamic simulated annealing to determine the structure. The structures computed had an average root-mean-square deviation about the mean structure of 0.41 angstrom for backbone atoms and 0.67 angstrom for heavy atoms when superimposed on the secondary structural elements.

Two-dimensional NMR 15N relaxation studies of the N-terminal SH3 domain of the protein drk indicate a stable and compact structure for the domain. This domain exists as an equilibrium between the folded and unfolded state of the protein in aqueous buffer. The variations in residue number induce changes in the spectral densities of the folded and unfolded states.

Analysis of backbone characteristics of phosphopeptide-complexed and free Src homology 2 domain reveals the link between large-scale restriction of picosecond-time-scale dynamics and peptide binding, caused by protein-protein interactions. NMR data reveal several cross peaks of protein residues in the binding site of SH2 complex.

Two- and three-dimensional double and triple resonance heteronuclear NMR techniques were used to obtain data related to 1H, 15N, 13C and 13CO assignments of the 19-kDa protein S to determine the proteins' three-dimensional structure. Protein S has the alpha-helix pattern which is distinct from other Greek key motifs. The Ca2+-binding protein S is a member of the beta gamma-crystalline family.

A study was conducted on the backbone dynamics of recombinant calmodulin using 2D nuclear magnetic resonance spectroscopy. Results suggested the flexibility of the region near the middle of the central helix of calmodulin. High degree of mobility was also observed in specific loops connecting calcium binding domains. The correlation times of the N- and C- terminal halves were also obtained and found to be different while compatible with the size disparity of their structured regions.

A study was conducted on the internal dynamics of methyl groups in proteins using nuclear magnetic resonance spectroscopy. The nuclear Overhauser effects and relaxation times of methyl carbons were measured based on the two-dimensional spectra obtained. The techniques were then applied to the 11 leucine side chains of staphylococcal nuclease (SNase). The internal motions of the leucine side chains were likewise obtained while a localized stiffening of the protein structure in regions near the ligand-binding sites was observed.

The application of antimalarial drug chloroquine as a new class of proteasome inhibitor that functions via a mechanism distinct from binding to active sites is reported. Choroquine is observed to inhibit proteasome function in eukaryotic cell extracts and is also found useful in preparation of purified 20S archaeal proteasome from Thermoplasma acidophilium.

The study results of [15.sup.N] relaxation dispersion of protein folding for a pair of SH3 domains, including the G48V mutant of a stabilized SH3 module from Abp1p and the A39V/N53P/V55L mutant of an SH3 domain from Fyn is presented. The results from the study suggest that formation of the intermediate state observed originally in studies of G48V and G48M Fyn SH3 might be a general feature of SH3 domain folding.

A detailed understanding of molecular recognition is predicated not only on high-resolution static structures of the free and bound states but also on information about how these structures change with time, that is, molecular dynamics. Here we present a deuterium (²H) and carbon (13C) NMR relaxation study of methyl side chain dynamics in the 82 kDa enzyme malate synthase G (MSG) that is a promising target for the development of new antibiotic agents. It is shown that excellent agreement between ²H- and 13C-derived measures of dynamics is obtained, with correlation coefficients exceeding 0.95. The binding interface formed by MSG and its substrates is found to be highly dynamic in the ligand-free state of the enzyme with rigidification upon binding substrate. This study establishes that detailed, quantitative information about methyl side chain dynamics can be obtained by NMR on proteins with molecular masses on the order of 100 kDa and opens up the possibilities for studies of motion in a large number of important systems. [ABSTRACT FROM AUTHOR]