Biomedical subjects

G Dodson

Publications and source records attributed to G Dodson.

At least 19 recordsLinked to original sources

PubMed2006

Protein flexibility: its role in structure and mechanism revealed by molecular simulations.

Computer simulations at the atomic level have arrived at a stage where they provide realistic modeling of flexibility in proteins (and the mobility of their associated solvent) that is important in understanding the nature of molecular motions. This can now be extended to the molecular and atomic motions that are associated with protein mechanisms. Moreover, the derived data agree reasonably accurately with experimental measurements of several kinetic and thermodynamic parameters. Fundamental insights emerge on the roles that this intrinsic flexibility plays in the thermodynamic characteristics of macromolecules in solution; these equip the investigator to probe the consequences of cognate interactions and ligand binding on entropy and enthalpy. Thus simulations can now provide a powerful tool for investigating protein mechanisms that complements the existing and the emerging experimental techniques.

Calmodulin↗

PubMed2004

Parity-violating electron deuteron scattering and the proton's neutral weak axial vector form factor.

We report on a new measurement of the parity-violating asymmetry in quasielastic electron scattering from the deuteron at backward angles at Q2=0.038 (GeV/c)2. This quantity provides a determination of the neutral weak axial vector form factor of the nucleon, which can potentially receive large electroweak corrections. The measured asymmetry A=-3.51+/-0.57 (stat)+/-0.58 (syst) ppm is consistent with theoretical predictions. We also report on updated results of the previous experiment at Q2=0.091 (GeV/c)2, which are also consistent with theoretical predictions.

Journal Article↗

PubMed2001

Crystal structure of the transcription elongation/anti-termination factor NusA from Mycobacterium tuberculosis at 1.7 A resolution.

Mycobacterium tuberculosis is the cause of tuberculosis in humans, a disease that affects over a one-third of the world's population. This slow-growing pathogen has only one ribosomal RNA operon, thus making its transcriptional apparatus a fundamentally interesting target for drug discovery. NusA binds to RNA polymerase and modulates several of the ribosomal RNA transcriptional processes. Here, we report the crystal structure of NusA, and reveal that the molecule consists of four domains. They are organised as two distinct entities. The N-terminal domain (residues 1 to 99) that resembles the B chain of the Rad50cd ATP binding cassette-ATPase (ABC-ATPase) and a C-terminal module (residues 108 to 329) consisting of a ribosomal S1 protein domain followed by two K homology domains. The S1 and KH domains are tightly integrated together to form an extensive RNA-binding structure, but are flexibly tethered to the N-terminal domain. The molecule's surfaces and architecture provide insights into RNA and polymerase interactions and the mechanism of pause site discrimination. They also allow us to rationalize certain termination-defective and cold shock-sensitive mutations in the nusA gene that have been studied in Escherichia coli.

Amino Acid Sequence↗

PubMed2001

Relativistic effects and two-body currents in (H)((-->)e(')p)n using out-of-plane detection.

Measurements of the (2)H((-->)e,e(')p)n reaction were performed with the out-of-plane magnetic spectrometers (OOPS) at the MIT-Bates Linear Accelerator. The longitudinal-transverse, f(LT) and f(')(LT), and the transverse-transverse, f(TT), interference responses at a missing momentum of 210 MeV/c were simultaneously extracted in the dip region at Q2 = 0.15 (GeV/c)(2). In comparison to models of deuteron electrodisintegration, the data clearly reveal strong effects of relativity and final-state interactions and the importance of two-body meson-exchange currents and isobar configurations. We demonstrate that such effects can be disentangled by extracting these responses using the novel out-of-plane technique.

Journal Article↗

PubMed2001

Crystallization and preliminary X-ray diffraction studies on the N-utilizing substance A (NusA) from Mycobacterium tuberculosis.

N-utilizing substance A (NusA) is a protein which performs several roles as a cofactor of DNA-dependent RNA polymerase. Its acts as an elongation factor and facilitates pausing, termination and the formation of a complex assembly that mediates transcription antitermination in eubacteria. Biochemical and biophysical data in the literature suggest that this protein performs these functions by binding to the core RNA polymerase, other protein factors and certain RNA fragments having specific signal sequences. The NusA of Mycobacterium tuberculosis has been cloned and overexpressed in Escherichia coli and crystallized using the hanging-drop vapour-diffusion method. The space group is P3(1)21, with unit-cell parameters a = b = 78.1, c = 180.3 A. A native data set complete to 1.7 A resolution has been collected from a single crystal.

Bacterial Proteins↗

PubMed2001

Search for quadrupole strength in the electroexcitation of the delta+(1232).

High-precision 1H(e,e'p)pi(0) measurements at Q2 = 0.126 (GeV/c)2 are reported, which allow the determination of quadrupole amplitudes in the gamma*N-->Delta transition; they simultaneously test the reliability of electroproduction models. The derived quadrupole-to-dipole ( I = 3/2) amplitude ratios, R(SM) = (-6.5+/-0.2(stat+sys)+/-2.5(mod))% and R(EM) = (-2.1+/-0.2(stat+sys)+/-2.0(mod))%, are dominated by model error. Previous R(SM) and R(EM) results should be reconsidered after the model uncertainties associated with the method of their extraction are taken into account.

Journal Article↗

PubMed2001

Spectroscopic and thermodynamic characterization of the transcription antitermination factor NusE and its interaction with NusB from Mycobacterium tuberculosis.

N-utilizing proteins (Nus) form a complex involved in the regulation of rRNA biosynthesis in enteric bacteria by modulating the efficiency of transcriptional termination [Nodwell, J. R., and Greenblatt, J. (1993) Cell 72, 261-268]. The protein NusE (identical to the protein S10 of the small ribosomal subunit) from the pathogenic mycobacterium M. tuberculosis has been cloned and overexpressed in Escherichia coli. The pure protein has been characterized by circular dichroism, ultracentrifugation, NMR, and binding to NusB. The near-ultraviolet circular dichroism spectrum of this protein suggests that it has a moderate (ca. 12-16%) alpha-helical content at 30 degrees C. The protein undergoes cold denaturation, with a temperature of maximum stability near 40 degrees C, implying a substantial heat capacity difference between the folded and unfolded states. The sedimentation equilibrium and velocity data indicate that the protein is monomeric and expanded in solution. NMR spectroscopy shows that there is no significant tertiary structure, and confirms the low secondary structure content at low temperatures. Furthermore, there was evidence for more structure at 30 degrees C than at 10 degrees C. Well-defined shifts in peaks in the HSQC spectrum of (15)N labeled NusE/NusB when the unlabeled counterpart was added at approximately stoichiometric concentrations showed the formation of a NusE-NusB complex in the absence of RNA. The far-UV CD and ultracentrifuge experiments, however, indicated relatively weak binding. Isothermal titration calorimetry showed the binding was weak and endothermic at 15 degrees C, with a total DeltaH of > or =10 kcal/mol. This weak binding is consistent with a small interaction interface and lack of large conformational rearrangements in the predominantly unfolded NusE protein. The conformational flexibility of NusE may be important for its roles in both the ribosome and antitermination complexes.

Amino Acid Sequence↗

PubMed2000

Strange magnetism and the anapole structure of the proton.

The violation of mirror symmetry in the weak force provides a powerful tool to study the internal structure of the proton. Experimental results have been obtained that address the role of strange quarks in generating nuclear magnetism. The measurement reported here provides an unambiguous constraint on strange quark contributions to the proton's magnetic moment through the electron-proton weak interaction. We also report evidence for the existence of a parity-violating electromagnetic effect known as the anapole moment of the proton. The proton's anapole moment is not yet well understood theoretically, but it could have important implications for precision weak interaction studies in atomic systems such as cesium.

Journal Article↗

PubMed2000

The molecular basis of vancomycin resistance in clinically relevant Enterococci: crystal structure of D-alanyl-D-lactate ligase (VanA).

d-alanine-d-lactate ligase from Enterococcus faecium BM4147 is directly responsible for the biosynthesis of alternate cell-wall precursors in bacteria, which are resistant to the glycopeptide antibiotic vancomycin. The crystal structure has been determined with data extending to 2.5-A resolution. This structure shows that the active site has unexpected interactions and is distinct from previous models for d-alanyl-d-lactate ligase mechanistic studies. It appears that the preference of the enzyme for lactate as a ligand over d-alanine could be mediated by electrostatic effects and/or a hydrogen-bonding network, which principally involve His-244. The structure of d-alanyl-d-lactate ligase provides a revised interpretation of the molecular events that lead to vancomycin resistance.

Bacterial Proteins↗

PubMed2000

Parity violation in elastic electron-proton scattering and the Proton's strange magnetic form factor

We report a new measurement of the parity-violating asymmetry in elastic electron scattering from the proton at backward scattering angles. This asymmetry is sensitive to the strange magnetic form factor of the proton as well as electroweak axial radiative corrections. The new measurement of A = -4.92+/-0.61+/-0.73 ppm provides a significant constraint on these quantities. The implications for the strange magnetic form factor are discussed in the context of theoretical estimates for the axial corrections.

Journal Article↗

PubMed2000

The crystal structure of NusB from Mycobacterium tuberculosis.

Both prokaryotes and eukaryotes regulate transcription through mechanisms that suppress termination signals. An antitermination mechanism was first characterized in bacteriophage lambda. Bacteria have analogous machinery that regulates ribosomal RNA transcription and employs host factors, called the N-utilizing (where N stands for the phage lambda N protein) substances (Nus), NusA, NusB, NusE and NusG. Here we report the crystal structure of NusB from Mycobacterium tuberculosis, the bacterium that causes tuberculosis in humans. This molecule shares a similar tertiary structure with the related Escherichia coli protein but adopts a different quaternary organization. We show that, unlike the E. coli homolog, M. tuberculosis NusB is dimeric both in solution and in the crystal. These data help provide a framework for understanding the structural and biological function of NusB in the prokaryotic transcriptional antitermination complex.

Amino Acid Motifs↗

PubMed1999

Structure of the trp RNA-binding attenuation protein, TRAP, bound to RNA.

The trp RNA-binding attenuation protein (TRAP) regulates expression of the tryptophan biosynthetic genes of several bacilli by binding single-stranded RNA. The binding sequence is composed of eleven triplet repeats, predominantly GAG, separated by two or three non-conserved nucleotides. Here we present the crystal structure of a complex of TRAP and a 53-base single-stranded RNA containing eleven GAG triplets, revealing that each triplet is accommodated in a binding pocket formed by beta-strands. In the complex, the RNA has an extended structure without any base-pairing and binds to the protein mostly by specific protein-base interactions. Eleven binding pockets on the circular TRAP 11-mer form a belt with a diameter of about 80 A. This simple but elegant mechanism of arresting the RNA segment by encircling it around a protein disk is applicable to both transcription, when TRAP binds the nascent RNA, and to translation, when TRAP binds the same sequence within a non-coding leader region of the messenger RNA.

Bacterial Proteins↗

PubMed1998

Bifunctional peptide boronate inhibitors of thrombin: crystallographic analysis of inhibition enhanced by linkage to an exosite 1 binding peptide.

The affinity of the hirudin49-64 segment for exosite 1 of thrombin has been used previously to enhance the potency of simple competitive inhibitors [DiMaio, J., Gibbs, B., Munn, D., Lefebvre, J. , Ni, F., Konishi, Y. (1990) J. Biol. Chem. 265, 21698-21703., and Maraganore, J. M., Bourdon, P., Jablonski, J., Ramachandran, K. L., and Fenton, J. W., II (1990) Biochemistry 29, 7095-7087.]. Using a similar approach, we have enhanced the activity of two active site directed thrombin inhibitors by attaching this segment via a novel reverse oriented linker to each of two tripeptide boronate inhibitors. At P1, compound 1 contains an arginine-like, isothiouronium, side chain, while compound 2 contains an uncharged, bromopropyl residue. Inhibition of human alpha-thrombin by compound 1 shows slow, tight-binding competitive kinetics (final Ki of 2.2 pM, k1 of 3.51 x 10(7) M-1 s-1, and k-1 of 1.81 x 10(-)4 s-1). The addition of hirugen peptide (20 microM) competes for exosite 1 binding and restores the k1 and k-1 to that of the analogous tripeptide, 0.29 x 10(7) M-1 s-1 and 0.13 x 10(-)4 s-1, respectively. Compound 1 has enhanced specificity for thrombin over trypsin with KiTry/KiThr of approximately 900 compared to the analogous tripeptide, with KiTry/KiThr of approximately 4. Compound 2 acts as a competitive inhibitor (KiThr of 0.6 nM) and is highly selective with no effect on trypsin. Crystallographic analysis of complexes of human alpha-thrombin with compound 1 (1.8 A) and compound 2 (1.85 A) shows a covalent bond between the boron of the inhibitor and Ser195 (bond lengths B-O of 1.55 and 1.61 A, respectively). The isothiouronium group of compound 1 forms bidentate interactions with Asp189. The P2 and P3 residues of the inhibitors form interactions with the S2 and S3 sites of thrombin similar to other D-Phe-Pro based inhibitors [Bode, W., Turk, D., and Karshikov, A. (1992) Protein Sci. 1, 426-471.]. The linker exits the active site cleft of thrombin forming no interactions, while the binding of Hir49-64 segment to exosite 1 is similar to that previously described for hirudin [Rydel, T. J., Tulinsky, A., and Bode, W. (1991) J. Mol. Biol. 221, 583-601.]. Because of the similarity of binding at each of these sites to that of the analogous peptides added alone, this approach may be used to improve the inhibitory activity of all types of active site directed thrombin inhibitors and may also be applicable to the design of inhibitors of other proteases.

Amino Acid Sequence↗

PubMed1998

The role of assembly in insulin's biosynthesis.

Insulin is synthesised as a single-chain precursor, preproinsulin, that contains an N-terminal signal sequence and a connecting peptide linking the A and B chains of the insulin molecule. Nascent proinsulin is directed into the regulated secretory pathway, converted to insulin and stored as microcrystals. These processes exploit assembly to the zinc-containing hexamer. Structural, chemical and genetic studies, and experiments with transgenic animals and transfected cells are providing new details about the molecular events in insulin's biosynthesis.

Animals↗

PubMed1998

Catalytic triads and their relatives.

Interactions among the residues in the serine protease Asp-His-Ser catalytic triad, in the special environment of the enzyme-substrate complex, activate the nucleophilic potential of the seryl O gamma. In the subtilisin and trypsin families, the composition and arrangement of the catalytic triad do not vary significantly. However, the mechanisms of action of many other hydrolytic enzymes, which target a wide range of substrates, involve nucleophilic attack by a serine (or threonine) residue. Review of these enzymes shows that the acid-base-ser/thr pattern of catalytic residues is generally conserved, although the individual acids and bases can vary. The variations in sequence and organization illustrate the adaptability shown by proteins in generating catalytic stereochemistry on different main-chain frameworks.

Amino Acid Sequence↗

PubMed1997

Tension in haemoglobin revealed by Fe-His(F8) bond rupture in the fully liganded T-state.

In 1972, Perutz proposed that the low affinity of T-state haemoglobin is caused by tension in the bond between the iron and the proximal histidine, restraining the Fe from moving into the porphyrin plane on binding oxygen. This proposal has often been disputed. If such tension does exist, it will be manifest in the liganded T-state. Here we describe the structure of the fully liganded T-state cyanide complex of haemoglobin, in which the Fe-proximal histidine bond in the alpha-subunits, but not in the beta-subunits, is ruptured. This rupture uncouples the structural changes at the alpha-haem from those in the globin and the beta-haem, and demonstrates unequivocally the existence of tension and its transmission through this bond.

Binding Sites↗

PubMed1997

The structure of the GTPase-activating domain from p50rhoGAP.

Members of the Rho family of small G proteins transduce signals from plasma-membrane receptors and control cell adhesion, motility and shape by actin cytoskeleton formation. They also activate other kinase cascades. Like all other GTPases, Rho proteins act as molecular switches, with an active GTP-bound form and an inactive GDP-bound form. The active conformation is promoted by guanine-nucleotide exchange factors, and the inactive state by GTPase-activating proteins (GAPs) which stimulate the intrinsic GTPase activity of small G proteins. Rho-specific GAP domains are found in a wide variety of large, multi-functional proteins. Here we report the crystal structure of an active 242-residue C-terminal fragment of human p50rhoGAP. The structure is an unusual arrangement of nine alpha-helices, the core of which includes a four-helix bundle. Residues conserved across the rhoGAP family are largely confined to one face of this bundle, which may be an interaction site for target G proteins. In particular, we propose that Arg 85 and Asn 194 are involved in binding G proteins and enhancing GTPase activity.

Amino Acid Sequence↗

PubMed1997

Determination of interspin distances between spin labels attached to insulin: comparison of electron paramagnetic resonance data with the X-ray structure.

A method was developed to determine the interspin distances of two or more nitroxide spin labels attached to specific sites in proteins. This method was applied to different conformations of spin-labeled insulins. The electron paramagnetic resonance (EPR) line broadening due to dipolar interaction is determined by fitting simulated EPR powder spectra to experimental data, measured at temperatures below 200 K to freeze the protein motion. The experimental spectra are composed of species with different relative nitroxide orientations and interspin distances because of the flexibility of the spin label side chain and the variety of conformational substates of proteins in frozen solution. Values for the average interspin distance and for the distance distribution width can be determined from the characteristics of the dipolar broadened line shape. The resulting interspin distances determined for crystallized insulins in the R6 and T6 structure agree nicely with structural data obtained by x-ray crystallography and by modeling of the spin-labeled samples. The EPR experiments reveal slight differences between crystal and frozen solution structures of the B-chain amino termini in the R6 and T6 states of hexameric insulins. The study of interspin distances between attached spin labels can be applied to obtain structural information on proteins under conditions where other methods like two-dimensional nuclear magnetic resonance spectroscopy or x-ray crystallography are not applicable.

Biophysical Phenomena↗