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Biomedical subjects

G W Vuister

Publications and source records attributed to G W Vuister.

At least 19 recordsLinked to original sources
PubMed2000

A novel experiment for the quantitative measurement of CSA(1H(N))/CSA(15N) cross-correlated relaxation in 15N-labeled proteins.

An experiment is presented which allows for the quantitative measurement of the relaxation interference between the 1H(N) CSA and 15N CSA interactions in 15N labeled proteins. A constant-time buildup scheme is used to measure the differential relaxation rate, eta, between double-quantum (DQ) and zero-quantum (ZQ) 1H(N)-15N coherences. The CSA/CSA experiment was recorded at three different Bo field strengths. The CSA(1H(N))/CSA(15N) cross-correlation rate was obtained from the linear fit of the measured rate, eta, versus Bo2 for 77 residues of the EH2 domain from mouse Eps15.

Adaptor Proteins, Signal Transducing↗
PubMed2000

The Mad1-Sin3B interaction involves a novel helical fold.

Sin3A or Sin3B are components of a corepressor complex that mediates repression by transcription factors such as the helix-loop-helix proteins Mad and Mxi. Members of the Mad/Mxi family of repressors play important roles in the transition between proliferation and differentiation by down-regulating the expression of genes that are activated by the proto-oncogene product Myc. Here, we report the solution structure of the second paired amphipathic helix (PAH) domain (PAH2) of Sin3B in complex with a peptide comprising the N-terminal region of Mad1. This complex exhibits a novel interaction fold for which we propose the name 'wedged helical bundle'. Four alpha-helices of PAH2 form a hydrophobic cleft that accommodates an amphipathic Mad1 alpha-helix. Our data further show that, upon binding Mad1, secondary structure elements of PAH2 are stabilized. The PAH2-Mad1 structure provides the basis for determining the principles of protein interaction and selectivity involving PAH domains.

Amino Acid Sequence↗
PubMed1999

The EH1 domain of Eps15 is structurally classified as a member of the S100 subclass of EF-hand-containing proteins.

The Eps15 homology (EH) domain is a protein-protein interaction module that binds to proteins containing the asparagine-proline-phenylalanine (NPF) or tryptophan/phenylalanine-tryptophan (W/FW) motif. EH domain-containing proteins serve important roles in signaling and processes connected to transport, protein sorting, and organization of subcellular structure. Here, we report the solution structure of the apo form of the EH1 domain of mouse Eps15, as determined by high-resolution multidimensional heteronuclear NMR spectroscopy. The polypeptide folds into six alpha-helices and a short antiparallel beta-sheet. Additionally, it contains a long, structured, topologically unique C-terminal loop. Helices 2-5 form two EF-hand motifs. Structural similarity and Ca(2+) binding properties lead to classification of the EH1 domain as a member of the S100 subclass of EF-hand-containing proteins, albeit with a unique set of interhelical angles. Binding studies using an eight-residue NPF-containing peptide derived from RAB, the cellular cofactor of the HIV Rev protein, show a hydrophobic peptide-binding pocket formed by conserved tryptophan and leucine residues.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Solution structure and backbone dynamics of the photoactive yellow protein.

The solution structure of photoactive yellow protein (PYP), a photosensory protein from Ectothiorhodospira halophila, has been determined by multidimensional NMR spectroscopy. The structure consists of an open, twisted, 6-stranded, antiparallel beta-sheet, which is flanked by four alpha-helices on both sides. The final set of 26 selected structures is well-defined for the regions spanning residues Phe6-Ala16, Asp24-Ala112, and Tyr118-Val125 and displays a root-mean-square deviation, versus the average, of 0.45 A for the backbone and 0.88 A for all heavy atoms. Comparison of the solution structure with an earlier published 1.4 A crystal structure (Borgstahl, G. E. O., Williams, D. R., and Getzoff, E. D. (1995) Biochemistry 34, 6278-6287) reveals a similarity with a root-mean-square deviation of 1.77 A for the backbone for the well-defined regions. The most distinct difference in the backbone with the crystal structure is found near the N-terminus, for residues Asp19-Leu23, which corresponds to an alpha-helix in the crystal structure and to one of the poorest defined regions in the solution structure. To characterize the dynamic behavior of PYP in solution, we undertook a 15N relaxation study and measurements of hydrogen/deuterium exchange. Determination of order parameters through the model-free Lipari-Szabo approach enabled the identification of several regions of enhanced dynamics. The comparison of atomic displacements in the backbone traces of the ensemble structures, with mobility measurements from NMR, show that the poorly defined regions feature fast internal motions in the nanosecond to picosecond time scale.

Bacterial Proteins↗
PubMed1998

Structural and dynamic changes of photoactive yellow protein during its photocycle in solution.

Light irradiation of photoactive yellow protein (PYP) induces a photocycle, in which red-shifted (pR) and blue-shifted (pB) intermediates have been characterized. An NMR study of the long-lived pB intermediate now reveals that it exhibits a large degree of disorder and exists as a family of multiple conformers that exchange on a millisecond time scale. This shows that the behavior of PYP in solution is different from what has been observed in the crystalline state. Furthermore, differential refolding to ground state pG is observed, whereby the central beta-sheet and parts of the helical structure are formed first and the region around the chromophore at a later stage.

Bacterial Proteins↗
PubMed1997

Heteronuclear NMR studies of the combined Src homology domains 2 and 3 of pp60 c-Src: effects of phosphopeptide binding.

The results of heteronuclear NMR studies on the combined Src homology domains 2 and 3 (SH3-SH2) of pp60 c-Src are presented. Resonance assignments were obtained using heteronuclear triple-resonance experiments in conjunction with 15N-separated nuclear Overhauser effect spectroscopy (NOESY) data. A modified three-dimensional 13CO-15N-1H spectral correlation experiment [(HACA)CO(CA)-NH] with improved sensitivity is presented that provided additional sequential information and resolved several ambiguities. Chemical shifts and sequential- and medium-range NOE cross peaks indicate that the structures of both the SH3 and SH2 portions of the polypeptide are very similar to those of the isolated SH3 and SH2 domains. Binding of a high-affinity phosphopeptide, EPQpYEEIPIYL, induces large chemical shift changes at several locations in the SH2 domain. Comparison with known results for peptide binding to SH2 domains shows that the residues displaying the largest effects are all involved in peptide binding or undergo significant conformational changes upon binding. However, subtle changes of both 1H and 15N chemical shifts are observed for residues within the SH3 domain and the connecting linker region, indicating possible cross-domain communication.

Amino Acid Sequence↗
PubMed1995

NMRPipe: a multidimensional spectral processing system based on UNIX pipes.

The NMRPipe system is a UNIX software environment of processing, graphics, and analysis tools designed to meet current routine and research-oriented multidimensional processing requirements, and to anticipate and accommodate future demands and developments. The system is based on UNIX pipes, which allow programs running simultaneously to exchange streams of data under user control. In an NMRPipe processing scheme, a stream of spectral data flows through a pipeline of processing programs, each of which performs one component of the overall scheme, such as Fourier transformation or linear prediction. Complete multidimensional processing schemes are constructed as simple UNIX shell scripts. The processing modules themselves maintain and exploit accurate records of data sizes, detection modes, and calibration information in all dimensions, so that schemes can be constructed without the need to explicitly define or anticipate data sizes or storage details of real and imaginary channels during processing. The asynchronous pipeline scheme provides other substantial advantages, including high flexibility, favorable processing speeds, choice of both all-in-memory and disk-bound processing, easy adaptation to different data formats, simpler software development and maintenance, and the ability to distribute processing tasks on multi-CPU computers and computer networks.

Magnetic Resonance Spectroscopy↗
PubMed1994

NMR evidence for similarities between the DNA-binding regions of Drosophila melanogaster heat shock factor and the helix-turn-helix and HNF-3/forkhead families of transcription factors.

Heteronuclear multidimensional NMR experiments of residues 33-163 of the DNA-binding domain of Drosophila heat shock factor, dHSF(33-163), were recorded, using only 3 mg of uniformly 15N-labeled or 2 mg of uniformly 15N/13C-labeled protein. The polypeptide consists of a structured part comprising three helices, a three-stranded antiparallel beta-sheet, with the first two strands connected by a four-residue type I tight turn. The second helix is disrupted at its C-terminal end by a proline residue and is followed by an extended turn, leading to the third helix. The dHSF(33-163) protein is unstructured at its N- and C-termini, and a third unstructured region is found from Thr113 to Arg124. Exchange broadening of the 15N-1H correlations upon titration of 15N labeled HSF with a 13-base-pair DNA duplex suggests a DNA-binding motif in which the third helix acts as the recognition helix. Both the secondary structure and DNA-binding pattern of dHSF(33-163) suggest that the overall topology resembles that the helix-turn-helix bacterial activator CAP [Weber, I. T., & Steitz, T. A. (1987) J. Mol. Biol. 198, 311-326] and the liver-specific transcription factor HNF-3 gamma, the prototype of the HNF-3/forkhead protein family [Clark, K. L., Halay, E. D., Lai, E., & Burley, S. K. (1993) Nature 364, 412-420].

Amino Acid Sequence↗
PubMed1994

Measurement of four-bond HN-H alpha J-couplings in staphylococcal nuclease.

Quantitative J-correlation and triple-resonance ECOSY-type experiments are used to unambiguously establish the presence of four-bond sequential HN-H alpha J-couplings in the protein staphylococcal nuclease. Substantially negative 4JH alpha HN values, ranging from -0.8 to -2.3 Hz, are observed when the psi angle is near +120 degrees, and the following phi angle near +60 degrees. For other conformations, the four-bond HN-H alpha J-couplings fall between -0.5 and +0.5 Hz.

Amino Acid Sequence↗
PubMed1994

Resonance assignment of methionine methyl groups and chi 3 angular information from long-range proton-carbon and carbon-carbon J correlation in a calmodulin-peptide complex.

Several simple 3D experiments are used to provide J correlations between methionine C epsilon methyl carbons and either the C gamma H2 protons or C beta and C gamma. The intensity of the J correlations provides information on the size of the three-bond J couplings and thereby on the chi 3 torsion angle. In addition, a simple 3D version of the HMBC experiment provides a sensitive link between the C epsilon H3 methyl protons and C gamma. The methods are demonstrated for a 20 kDa complex between calmodulin and a 26-residue peptide fragment of skeletal muscle myosin light chain kinase.

Amino Acid Sequence↗