Search

New labeling approach and NMR experiments are described along with those that has probed the exchange by using [super 15]N and [super 1]H N nuclei. The experimental data has showed that for many systems accurate excited-state chemical shifts can be measured, thus opening a possibility of obtaining quantitative structural information of invisible states of the sort that is recorded on ground states of proteins.

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.

The pressure dependence of (super 15)N NMR relaxation dispersion rate profiles were used to study the changes in volumetric parameters that accompany the folding reaction of protein at 45 degree C. The results argue for a transition state ensemble (TSE) that is collapsed but loosely packed relative to the folded state and significantly hydrated, suggesting that the release of water occurs after the rate-limiting step in protein folding.

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.

An approach for four-dimensional (4D) recording of methyl (super 1)H-(super 13)C-(super 1)H NOESY spectra with high resolution and sensitivity is presented and applied to Malate Synthase G (MSG). The methyl-methyl distance restraints in the present study are critical to the success of structural studies of MSG.

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.

Near complete stereospecific assignments of the prochiral methyl carbons of Leu and Val residues in malate synthase G, a 723 residue enzyme, are reported. Assignments were obtained on the basis of a 10 percent fractional (super 13)C-labeling, strategy developed by Wuthrich and co-workers and, in case of Val residues, supplemented with results from a series of new methyl-TROSY quantitative J experiments.

The assignment of methyl (super 13)C and (super 1)H chemical shifts from Ile, Leu, and Val residues in high molecular weight proteins, and it is studied using new NMR experiments. Results show that the size of the proteins increases significantly and highlights the possibility of using side-chain methyl groups for probing the molecular structure and dynamics in high molecular weight systems.

Residual interactions in delta131delta, a large disordered fragment of staphylococcal nuclease is probed at two different pHs using backbone (super 15)N and side-chain methyl (super 2)H NMR spin relaxation techniques. Dramatic increases in the amplitudes of motions occurring on a nanosecond time scale are seen throughout delta131delta at pH 3 relative to pH 5.

A sensitive 3D NMR pulse scheme, (H)C(CA)NH-COSY is presented for the assignment of 13C(super delta1) IIE chemical shifts in large perdeuterated, methyl protonated proteins. The experiment presented utilizes COZY-based transfer steps and refocuses undesirable 13C-13C scalar couplings that degrade the efficiency of TOCSY transfers.

A new set of pulse schemes for the study of methyl-containing side chain dynamics in partially folded and unfolded protein states is presented. Methyl side chain dynamics in an unfolded protein deletion mutant, Delta 131 Delta is studied.

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.

A number of simple and sensitive experiments for measuring the sign of delta omega in systems with millisecond-time scale exchange, which are best suited for analysis, using CPMG-based dispersion measurements are presented. The application of the methodology involving C(super 13) NMR, where chemical shifts can be very sensitive to secondary structure, will shed further light on the structural features of the excited state of the protein.

Numerous carbohydrate-processing enzymes facilitate catalysis via stabilization of positive charges near the C1, C4, C5 or C6 positions, but reactions catalyzed by these enzymes are diverse. An easy method that allows easy access to different varieties of 5-fluoro group glycosides as well as glycosyl phosphates is developed with the aim to solve the difficulties faced in introducing the 5-fluoro substituent after radical halogenation.

The measurement of the five different quadrupolar deuterium relaxation rates associated with a single spin-1 particle is demonstrated to carry out detail analysis of protein side-chain dynamics, including the identification of nanosecond-time-scale motions. The results of a comprehensive spectral density mapping study of side-chain methyl deuterons in proteins are reported.

The measurement of the relaxation of deuterium double quantum, quadrupolar order and transverse antiphase magnetization in 13CH2D methyl groups of 15N-, 13C-labeled, fractionally deuterated proteins is reported. These schemes allow measurement of the five unique decay constants of a single deuteron, providing an aid to examine side-chain dynamics in proteins.

The study presents a report on the triple nuclear magnetic resonance pulse scheme, which is useful in measuring aspartic and glutamic acid side-chains. The study correlates side-chain carboxyl carbon chemical shifts in these residues.

The CPMG-based relaxation dispersion techniques used to analyze the conformational exchange processes in the folded and unfolded [F(sub exch)/U(sub exch)] system of the drkN SH3 domain is described. The results show that relaxation dispersion experiments give an extremely sensitive tool to probe conformational exchange processes in unfolded states and to get information on the free energy landscape of such systems.

The use of a short, three-residue Cu (super 2+) -binding sequence, the ATCUN motif, is presented as an approach for extracting long-range distance restraints from relaxation enhancement NMR spectroscopy. The ATCUN motif is prepended to the N -termini of proteins and binds Cu (super 2+) with a very high affinity.

The measurement of mu-s-ms time scale dynamics of Asn and Gln side chains in proteins is discussed. Many of the side chain amide groups of Asn and Gln residues in the C-terminal domain of the protein were shown to be influenced by a chemical exchange process that could be significant in allowing the rapid binding of hydrophobic ligands to the cavity.

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

The uniformity of the methyl 2H quadrupolar coupling in oriented proteins is considered.

A study was conducted to analyze the TROSY triple resonance four-dimensional nuclear magnetic resonance (NMR) spectroscopy of a 46 ns tumbling protein. In line with this, spectra were determined on a 1.4 mM sample of 15N, 13C and 2H using the Varian Inova 600 MHz spectrometer. Results indicated the effectiveness of four-dimensional TROSY-based triple-resonance NMR spectroscopy in determining protein backbone assignments.

A triple resonance nuclear magnetic resonance approach for measuring protein backbone dihedral angle psi is presented. The approach is based on the cross-correlation spin relaxation between C(super alpha)-H(super alpha) and 15N-HN dipolar interactions in 15N,13C-labeled proteins. Results show that the possible psi value deceases from four to two in a combination of cross-correlation relaxation rates. A single value can be derived for psi values ranging from -50 degrees to 40 degrees.

A three-dimensional NMR experiment for measuring methyl-methyl NOEs in 13C-labeled proteins with high resolution is conducted. The method is well suited for studies of fully protonated proteins, as shown by an application involving the 160 residue phosphotyrosine binding domain from Drosophila Numb. Results indicate that distance restraints obtained from the study are very useful in the production of global protein folds.

A pulse scheme is developed and used for recording nuclear Overhauser effects (NOEs) from valine and isoleucine methyl groups to proximal protons. The method offers significantly improved resolution in comparison with other 13C-edited experiments because both the indirectly identified dimensions are recorded in constant-time mode. The utility of the pulse scheme can be seen with applications on a fully protonated 15N,13C-labeled sample and a methyl protonated, 15N,13C,2H-labeled complex of 42 kDa.

Intermolecular NOEs in a molecular complex comprising super15N, super13C labeled and unlabeled components were recorded, using new pulse schemes. Adiabatic super13C inversion pulses were used for filtering. Improved spectra was obtained relative to data from other purging schemes. Structural information was obtained for the amino-terminal domain of the N protein from bacteriophage lambda and boxB RNA, using the new scheme.

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.

The almost complete (>90%) NMR assignment of 15N, 13C-alpha, 13C-beta, and HN chemical shifts is shown for a 64 kDa trp repressor-operator complex composed of two tandem dimers of 15N, 13C, >90% 2H labeled trp repressor, unlabeled 22-base-pair DNA, and unlabeled corepressor, 5-methyltryptophan. Constant-time chemical shift evolution of the carbon magnetization are used, resulting in substantial enhancements in spectral resolution compared to non-constant-time versions of the pulse schemes.

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.

The dynamics of methyl groups are measured by calculating the 2H T(sub 1) and T(sub 1-rho) relaxation times in proteins which are uniformly C(super 13) labeled and partially deuterated. This new method of measurement is based on a constant-time H(super 1)-C(super 13) HSQC sequences, and requires mathematical interpretation. The relaxation nature of CH2D methyl groups establish that relaxation rates can be measured accurately. The sidechain dynamics of the C-terminal SH2 domain from phospholipase C(sub gamma-1) are also determined using the new method.

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.

Various triple resonance NMR experimental schemes, based on a principle that substitution of deuterons for bound protons leads to the line narrowing of the 13C spins, help accurate backbone assignments in 15N, 13C, 2H labeled proteins. Application of these methods on a 37kDa ternary complex of 15N, 13C and approximately 70% 2H labeled trp-repressor, 5-methyltryptophan and a 20 basepair trp-operator DNA fragment helps determine intra and/or inter-residue correlations. Deuteration enhances the average 13C alpha T2 value, as revealed by carbon relaxation data of the complex.

Sensitive two-dimensional NMR studies help determine 13(alpha) dynamics in highly-enriched, uniformly 13C-labeled proteins at various temperatures. The neighboring carbon spins influence carbon relaxation properties in 13C(alpha)-alanine and 13(3) alanine. Theeoretical studies suggest the possibility of accurate measurement of 13C(alpha)T(1P) values for all residues except serine and threoine, which have equal 13C(alpha)and 13C(beta) chemical shifts.

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.