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The transient, ultra-weak self-association, involving the histidine-containing protein HPr by means of paramagnetic relaxation enhancement (PRE), is detected and characterized by using EDTA-[Mn.sup.2+] conjugated at three separate sites. Large intermolecular PRE effects are seen for two of the three paramagnetically labeled sites, E5C and E32C.

A carbon-detected triple resonance experiment, c-TROSY-HNCO, is described with transverse relaxation-optimized spectroscopy (TROSY) optimization in all dimensions and sensitivity enhancement in both indirect directions. A new, sensitivity enhancement method, called double echo-antiecho (dEA), is also detailed that has provided sensitivity enhancement of [square root of 2] in both indirect dimensions.

A series of heteronuclear NMR experiments for the direct observation and characterization of lysine [NH.sub.3] groups in proteins is studied. Results reveal that the [super 15]N relaxation for antiphase coherence with respect to [super 1]H through scalar relaxation of the second kind.

A-box of the classical nonspecific DNA binding protein HMGB-1 (HMGB-1A) is used as an example to sow that H-PRE measurements also offer a powerful and unique approach for studying large-scale dynamics associated with nonspecific protein-DNA interactions. The intermolecular 1H-PRE data demonstrate unambiguously that HMGB-1A binds to multiple sites in multiple orientations even on a DNA fragment as short as 14 base pairs.

The (super 1) H(sub N)/(super 15) N chemical shift mapping data are transformed into a set of highly ambiguous, intermolecular distance restrains (comprising between 400 and 3000 individual distances) with transnational and some degree of orientational information content, while the dipolar couplings provide information on relative protein-protein orientation. The methodology presented should greatly accelerate the determination of higher accuracy NMR structures of complexes (including the detailed placement of interfacila side chains) by providing a good starting point for the assignment of intermolecular NOE data.

Research is presented describing the study of cyanovirin-N domain-swapped dimer to determine molecular orientation.

The determination of macromolecular structures using nuclear magnetic resonance spectroscopy is a minimization problem that involves fitting atomic models to observable experiments. An expression for the dipolar coupling R-factor that is capable of providing a readily understandable measure of the relationship between observed and calculated dipolar coupling values has been derived. The dipolar R-factor, which features complete cross-validation, is applied to data derived for the cyanovirin-N protein.

The establishment of a quantitative connection between backbone-backbone hydrogen-bond (H-bond) length seen in protein crystal structure and (super 3h)J(sub NC') couplings across such bonds is limited by the coordinate precision of the X-ray structure. Very high-resolution X-structures are available for an immunoglobulin binding domain of streptococcal protein G. Findings showed that over the small range of N-O H-bond lengths for which (super 3h)J(sub NC') couplings are observable, the 32 measured (super 3h)J(sub NC') values can be fit to: (super 3h)J(sub NC') = -59000 exp(-4R(sub NO)) +/- 0.09 Hz, or R(sub NO) = 2.75 - 0.25 ln(-(super 3h)J(sub NC')) +/- 0.06 angstrom.

The interaction between the 30 kilodalton amino-terminal domain of enzyme I (EIN) and the histidine-containing phosphocarrier protein HPr was analyzed by nuclear magnetic resonance (NMR). NMR analysis of the association constant between EIN and HPr indicated the important role of the H2 and H2prime helices in the function of EI. Furthermore, contacts between HPr and EIN occurred between the N-terminal end of the interacting helix of HPr and helix H2 of the domain alpha including the loop connecting the beta3 strand and helix H6 of the alpha/beta domain of EIN.

Nuclear magnetic resonance spectroscopy was done to determine the three-dimensional solution structure of the 259-residue 30 kDa N-terminal domain of enzyme I (EIN) of the phosphoenolpyruvate:sugar phosphotransferase system derived from Escherichia coli. The results showed that the conformation of the active site His189 is locked in a single trans chi-1 rotamer conformation with he N-epsilon-2 atom directed toward the O-gamma-1 atom of the Thr168. It was also found that the neutral form of His189 is the N-delta-1-H tautomer with the N-epsilon-2 atom of His189 forming a hydrogen bond with the O-gamma-H hydroxyl proton of Thr168.

Mammalian male sex determination is controlled by a complex hierarchy of gene regulatory proteins and hormones, which promote male gonadal development and regression of the female primordia. At the core of this pathway lies the SRY protein, the master developmental switch for testicular differentiation and hence, the male sex. The three-dimensional structure of the SRY-DNA complex suggests a model of developmental gene regulation in which proteins that alter DNA structure and promote the assembly of higher-order nucleoprotein complexes play an essential role in the timing of cell specialization events.

Multidimensional triple-resonance nuclear magnetic resonance spectroscopy techniques were used to determine the ionization constant (pKa) of side-chain carboxyl groups of human thioredoxin in oxidized and reduced form as well as in complex with the target peptide from transcription factor NF-kappa-B. The results show that thioredoxin has a pKa of 9.9, 8.1 and 8.9 in reduced form, oxidized form and mixed disulfide complex, respectively.

The complex of an eight base pair DNA oligonucleotide duplex with the human testis determining factor SRY determines the association of DNA protons to C(super 12) in the presence of proportionally surplus C(super 13)/N(super 15) protein when analyzed by NMR methods. In SRY-bound DNA, the DNA is underwound and bent through expansion of the minor groove and contraction of the major group. The SRY-bound DNA has different conformation than the A-DNA or B-DNA. In the DNA octamer, partial intercalation of a protein drain among two adenine bases is identified.

The DNA binding domain of the integrase protein of the human immunodeficiency virus in solution exists in the form of a dimer. Each subunit is formed of a five-stranded beta-barrel whose structure is similar to that of the SH3 domain. There is a stacked beta interface and the each subunits' triple stranded antiparalled beta-sheets is antiparallel to the other. The surface bound by beta 1 and 2 strands forms the saddle shaped groove to which the DNA binds. Integrase is involved in the integration of the viral genome into the host chromosome.

Examination of the binding of purified phage Mu Ner protein to various DNA fragments of different lengths, reveals that decrease in the size of the DNA fragment weakens the binding between DNA and the Mu Ner protein. A modified Ferguson method helps determine the stoichiometry of Ner complexes with the 30 and 18 bp DNA fragments. Experimental data reveal the simultaneous binding of two Ner monomers to DNA. A proposed model explains the binding between DNA and Ner.

Gibbs energy of a hydrogen bond decreases with an increase in hydrogen bond length and the number of helices. Study of the stability of interleukin-1-beta in comparison with other globular proteins reveals that the Gibbs energy of hydrogen bond in a beta-sheet structure is much higher than that of alpha-helices. Interleukin-1-beta changes from a native to a denatured state with rise in temperature. Transition, which occurs with significant heat absorption, is irreversible above pH 3.5.

Multidimensional heteronuclear nuclear magnetic resonance spectroscopy yields the folding topology and secondary structure of the first RNA binding domain of the hnRNP A1 protein. A beta-alpha-beta-beta-alpha-beta folding pattern is exhibited by amino acid long chains, which are arranged in four-stranded antiparallel beta-sheet supported by two alpha-helices. The loops which attach the secondary structural elements transform the binding proteins of the structurally-classified RNA.

The 3-D structure of interleukin-4 (IL-4) is characterized by a left-handed four-stranded helix interlinked by long loops and by short strands of small antiparallel beta sheet or helical turns. The tight packing of the helices results in backbone atomic rms difference of 1.37 Angstrom, as illustrated by the superposition of the NMR structure of IC-4 with the 2.25 Angstrom unit crystal structure. IL-4 belongs to the cytokine family of proteins and contribute to the activities in the immune and inflammatory systems.

A study was conducted on the structure of the B2 immunoglobulin binding domain of streptococcal protein G determined at 1.67 angstrom resolution. Results were compared with resonance measurements of the B1 domain structure. The existence of 83 residues was revealed and these residues were found to have an average B factor of 18. Both the B1 and B2 domains were also found to be comprised of four beta-strands and one helix lying diagonally across the beta-sheet.

Two-dimensional inverse-detected heteronuclear 15N-1H nuclear magnetic resonance spectroscopy was used to study the backbone dynamics of 15N-labeled ribonuclease H (RNase) domain of human immunodeficiency virus (HIV-1) reverse transcriptase. The RNase H domain was found to exhibit mobility throughout its structure. In addition, the C-terminal residues from Tyr-110 to Leu138 and the loops between beta-strands beta1 and beta2 and between alpha-helix alphaB and beta-strand beta4 showed increased overall mobility relative to the remainder of the protein. These results suggest a substrate binding site conformation that explains the lack of enzymatic activity of RNase H domain.

The determination of the secondary structure, topology and global fold of human recombinant interleukin-4 (IL-4) was presented. Based on an analysis of three-dimensional heteronuclear-edited Overhauser spectra, 13C chemical shift, amide chemical exchange and (super 3)J(sub HNalpha) coupling constant data, it was shown that IL-4 consists of four long helices, two small helical turns and a mini antiparallel beta-sheet. In addition, the helices are arranged as a left-handed four-helix bundle with two overhand connections.

The 1H, 15N, 13C and 13CO assignments of the spectrum of recombinant human interleukin-4, a 15.4-kDa protein of 133 residues, were presented. A series of three-dimensional double- and triple-resonance nuclear magnetic resonance experiments was used to correlate 1H, 15N, 13C and 13CO chemical shifts by means of large intra- and sequential interresidue heteronuclear scalar couplings.

Nuclear magnetic resonance spectroscopy was used to elucidate the high-resolution three-dimensional solution structure of synthetic 57-residue double zinc finger of the major histocompatibility complex enhancer binding protein (MBP-1) of humans. The results suggest that there are different contacts involved in the binding of the double Cys2His2 zinc fingers of MBP-1.

The three-dimensional solution structure of a dihydrolipoamide dehydrogenase-binding domain from a 2-oxo acid dehydrogenase multienzyme complex of Escherichia coli was determined by nuclear magnetic resonce spectroscopy and hybrid distance geometry-dynamical simulated annealing calculations. The structure is built on 630 interproton distance and 101 torsion angle restraints and has two parallel helices, a short extended strand,a five-residue helical-like turn, and a highly disorganized loop.

The mechanism of redox function and the electrostatic behavior of human thioredoxin was evaluated with the use of two-dimensional nuclear magnetic spectroscopy. A total of 241 titration curves were measured over a pH range from a predertermined spectra. Results revealed a complex behavior caused by interacting titrating groups, protein interior electrostatic interactions and probably by conformational changes on the chemical shifts.

An NMR approach to directly determine the distribution radius of a disordered group independent of any structural knowledge is described. The results demonstrated the application of developed formalism for paramagnetic relaxation enhancement vectors and for [super 1]H-[super 1]H cross-relaxation rates which could be used as an alternative approach for analyzing the breadth of the positional distribution of disordered groups.

Residual dipolar couplings and small- and wide-angle X-ray scattering are used to solve the solution structures of free 128 kDa Enzyme I (EI), the first enzyme in the bacterial phosphotransferase system and its 146 kDa complex with HPr. The large scale interdomain motins observed for the free EI and the EI-HPr complex when compared to that of the crystal structure of a trapped phosphorylated EI intermediate offer insight into the structural transitions that accompany the catalytic cycle of EI.

NMR relaxation measurements are used for addressing the kinetics of exchange between monomeric and large, polymorphic oligomeric species of amyloid [beta] (A[beta](1-40)). The increase in [super 15]N and [super 1][H.sub.N][R.sub.2] rates as a function of protein concentration is independent of nucleus and magnetic field and the fraction of peptide within the dark oligomeric state undergoing exchange with free monomer is calculated.

A pseudopotential [E.sub.relax] is described that permits direct use of [super 15]N relaxation rates in the form of [R.sub.2]/[R.sub.1] ratios as experimental restraints in structure calculations without needing prior information to be extracted from a known molecular structure. The [E.sub.relax] term is used for protein-protein docking of complexes and has shown its applicability to the 40 kDa complex of the N-terminal domain of enzyme I and the histidine phosphocarrier protein HPr and to the symmetric HIV-1 protease dimer.

An approach is described for making use of the components of the experimentally determined rotational diffusion tensor derived from NMR relaxation measurements in macromolecular structure determination. The studies have shown the utility of rotational diffusion tensor restrains for protein structure refinement by using the N-terminal domain of enzyme I (EIN) as an example.

The study demonstrates the importance of a TROSY-based-z-exchange experiment in determining the activation energy for intermolecular protein translocation between the specific sites on different DNA molecules. The process can also be easily applied at extremely low temperatures.

The complex of the N-terminal domain of enzyme I (EIN) and H[Pr.sup.4.7] is used for examining the ionic strength dependence of intermolecular paramagnetic relaxation enhancement (PREs). The study of complex proves that the interactions involved in the formation of short-lived encounter complexes are electrostatic in nature.

Conformational changes in gp120-gp41 expressed on cells as a function of time following interactions of Env-expressing cells with target cells using a nonspecific probe is monitored using analytical and quantitative video microscopy. The reactivity of triggered gp41 to the NC-1 monoclonal antibody, which is demonstrated to bind to N-helical gp41, increased rapidly to a maximal level in the primed state but decreased once stable fusion junctions had formed.

Backbone residual dipolar coupling (N-H, C alpha-H alpha, N-C', and C alpha-C') data collected in five different media on the B3 IgG binding domain of streptococcal protein G(GB3) are analyzed. This process is done through simultaneous refinement of the coordinates and optimization of the magnitudes, and orientations of the alignment tensors using single and multiple structure representations.

A new approach that aids in the calculation of structures is presented. This approach has a backbone accuracy of 1.0-1.5 angstrom taken from databases in which as much as 80% of the long-range overhauser enhancement information is incorrect.

A theoretical and computational approach to make use of (super 1)H paramagnetic relaxation enhancement (PRE) data arising from a flexible paramagnetic group, as a source of restraints for NMR structure calculations, is presented. The importance of taking into account the conformational space resembled by the flexible paramagnetic group is showed.

A new approach for deriving kinetic information on protein-DNA interactions by NMR is applied o the interaction of the human Hox-D9 homeodomain with DNA, which highlights the mechanism of rapid protein translocation between cognate DNA binding sites and reconciles the long half-lives of specific protein. The translocation of the whole protein from one oligonucleotide to the other might contribute to rapid specific target search through nonspecific protein-DNA interactions, as the concentration of nonspecific sites is enormous.

EDTA-derivatized deoxythymidine is presented as a tool for rapid determination of protein binding polarity to DNA by intermolecular paramagnetic relaxation. The method is evaluated and it is found that it has widespread utility in structural and chemical studies of protein-DNA complexes.

The study has shown the equilibrium side chain X1 angles and rotamer distributions which are predicted from static crystal structures using a torsion angle database potential of mean force in conjunction with rigid body angle dynamics. For a mobile surface side chain exhibiting rotamer averaging, the equilibrium distributions of rotamers will largely be determined by its backbone.

A method, based on dipolar couplings, for identifying medium- to large-scale interdomain motion in molecular proteins is described.

A simple, dipolar couplings-based approach for obtaining accurate orientations of the functional groups of asparagine and glutamine side chains is presented.

The inclusion of a pseudopotential for the radius of gyration, R(sub gyr), into NMR structure calculations has been investigated. The study seeks to determine if the pseudopotential can significantly increase the accuracy of computations particularly in protein-protein complexes. The R(sub gyr) is defined as the rms distance from each atom of the molecule to their centroid. The computed values of R(sub gyr) can be verified experimentally through small-angle x-ray scattering spectroscopy.

In measurements of residual dipolar couplings, the macromolecule of interest must by weakly aligned in the magnetic field. Alignment can be induced by the magnetic field itself or using a liquid crystalline medium. Macromolecules dissolved in dilute liquid crystalline phase of lipid bicelles can achieve moderate degrees of alignment. The bicelles are thermotropic and adopt a liquid crystalline phase over narrow temperature ranges. The nematic phase of a colloidal suspension of rod-shaped virusues is shown to provide an alternative to bicelles.

A simple procedure is described for obtaining the magnitude of the fully asymmetric diffusion tensor from the distribution of heteronuclear T1/T2 ratios in the absence of structural information. Not only information regarding the shape and hydrodynamic properties of the macromolecule under consideration can be derived from this method. It also allows the use of heteronuclear T1/T2 ratios for structure refinement.

Research into methods of increasing the accuracy of DNA structure analysis through NMR is presented. The paper proposes the use of a database potential of mean force which offers a statistical description of base-base positions.

Research was conducted to examine the impact of residual dipolar couplings on the accuracy of NMR structures determined from a minimal number of NOE restraints. The objective was to show that the use of dipolar couplings leads to large improvements in accuracy even when minimal NOE data sets are used. Results demonstrate that the inclusion of dipolar couplings in structure calculations can lead to substantial increases in accuracy even for data sets consisting of a minimal number of NOE restraints.