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R Kaptein

Publications and source records attributed to R Kaptein.

At least 37 records · Page 2Linked to original sources
PubMed1999

Editing of chemical exchange-relayed NOEs in NMR experiments for the observation of protein-water interactions.

An experimental approach for the editing of exchange-relayed NOEs in water-selective NOE experiments is presented. The proposed pulse sequence is based on the application during the NOE mixing time of continuous wave irradiation, which saturates resonances of relaying labile protons in slow chemical exchange with water. The technique can efficiently reduce the contributions of exchange-relayed NOE peaks that often crowd the water-selective NOE spectra and hide direct intermolecular NOEs between water and protein protons. The present approach opens new opportunities for the characterization of hydration by NMR, even in the proximity of polar labile groups.

Macromolecular Substances↗
PubMed1999

Water-macromolecule interactions by NMR: a quadrature-free constant-time approach and its application to CI2.

A pulse sequence is proposed to select water magnetization with enhanced specificity through a synergetic combination of several filtering principles. This approach relies on a constant-time evolution period implemented without quadrature detection, which results in a square root 2 increase in signal-to-noise ratios as compared to traditional non-selective methods for water filtration. In addition, the quadrature-free constant-time block facilitates the implementation of the water flip-back strategy, which leads to further gains in sensitivity. The proposed experiment was applied to unlabeled HEW lysozyme and to 15N-labeled chymotrypsin inhibitor 2 which was partially or non 13C-enriched. Water molecules belonging to a spine of hydration between two pseudo beta-sheet strands were identified, solving previously reported discrepancies between the X-ray and refined NMR structure of CI2. The proposed experiment in particularly suitable for hydration studies of mixtures of labeled and unlabeled components, such as ligand-macromolecule complexes.

Hydrogen Bonding↗
PubMed1999

Completeness of NOEs in protein structure: a statistical analysis of NMR.

The completeness of experimentally observed NOE restraints of a set of 97 NMR protein structures deposited in the PDB has been assessed. Completeness is defined as the ratio of the number of experimentally observed NOEs and the number of 'expected NOEs'. A practical definition of 'expected NOEs' based on inter-proton distances in the structures up to a given cut-off distance is proposed. The average completeness for the set of 97 structures is 68, 48, and 26% up to 3, 4, and 5 A cut-off distances, respectively. For recent state-of-the-art structures these numbers are approximately 90, 75, and 45%. Almost 20% of the observed NOEs are between atoms that are further than 5 A apart in the final structures. The completeness is independent of the relative surface accessibility and does not depend strongly on residue type, secondary structure or local precision, although the number of observed NOEs in these classes varies considerably. The completeness of NOE restraints is a useful quality criterion in the course of structure refinement. The completeness per residue is more informative than the number of NOEs per residue, which makes it a useful tool to assess the quality of the NMR data set in relation to the resulting structures.

Amino Acid Sequence↗
PubMed1999

Microsecond time scale dynamics in the RXR DNA-binding domain from a combination of spin-echo and off-resonance rotating frame relaxation measurements.

Slow protein dynamics can be studied by 15N spin-echo (CPMG) and off-resonance rotating frame relaxation through the effective field dependence of the exchange-mediated relaxation contribution. It is shown that, by a combination of these complementary techniques, a more extended sampling of the microsecond time scale processes is achieved than by either method alone. 15N R2 and improved off-resonance R1 rho experiments [Mulder et al. (1998) J. Magn. Reson., 131, 351-357] were applied to the 9-cis-retinoic acid receptor DNA-binding domain and allowed the identification of 14 residues exhibiting microsecond time scale dynamics. Assuming exchange between two conformational substates, average lifetimes ranging from 37 to 416 microseconds, and chemical shift differences of up to 3 ppm were obtained. The largest perturbation of tertiary structure was observed for the second zinc finger region, which was found to be disordered in the solution structure [Holmbeck et al. (1998) J. Mol. Biol., 281, 271-284]. Since this zinc-coordinating domain comprises the principal dimerization interface for RXR in a wide repertoire of complexes with different hormone receptors to their cognate response elements, this finding has important implications for our understanding of nuclear receptor assembly on DNA direct repeats. The flexibility observed for the dimerization domain may explain how RXR, through the ability to adaptively interact with a wide variety of highly homologous partner molecules, demonstrates such a versatile DNA-binding repertoire.

Amino Acid Sequence↗
PubMed1999

The tetramerization domain of the Mnt repressor consists of two right-handed coiled coils.

The tetrameric Mnt repressor is involved in the genetic switch between the lysogenic and lytic growth of Salmonella bacteriophage P22. The solution structure of its C-terminal tetramerization domain, which holds together the two dimeric DNA-binding domains, has been determined by NMR spectroscopy. This structure reveals an assembly of four alpha-helical subunits, consisting of a dimer of two antiparallel coiled coils with a unique right-handed twist. The superhelical winding is considerably stronger and the interhelical separation closer than those found in the well-known left-handed coiled coils in fibrous proteins and leucine zippers. An unusual asymmetry arises between the two monomers that comprise one right-handed coiled coil. A difference in the packing to the adjacent monomer of the other coiled coil occurs with an offset of two helical turns. The two asymmetric monomers within each coiled coil interconvert on a time scale of seconds. Both with respect to symmetry and handedness of helical packing, the C2 symmetric four-helix bundle of Mnt differs from other oligomerization domains that assemble DNA-binding modules, such as that in the tumor suppressor p53 and the E. coli lac repressor.

Amino Acid Sequence↗
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

Recommendations for the presentation of NMR structures of proteins and nucleic acids--IUPAC-IUBMB-IUPAB Inter-Union Task Group on the standardization of data bases of protein and nucleic acid structures determined by NMR spectroscopy.

The recommendations presented here are designed to support easier communication of NMR data and NMR structures of proteins and nucleic acids through unified nomenclature and reporting standards. Much of this document pertains to the reporting of data in journal articles; however, in the interest of the future development of structural biology, it is desirable that the bulk of the reported information be stored in computer-accessible form and be freely accessible to the scientific community in standardized formats for data exchange. These recommendations stem from an IUPAC-IUBMB-IUPAB inter-union venture with the direct involvement of ICSU and CODATA. The Task Group has reviewed previous formal recommendations and has extended them in the light of more recent developments in the field of biomolecular NMR spectroscopy. Drafts of the recommendations presented here have been examined critically by more than 50 specialists in the field and have gone through two rounds of extensive modification to incorporate suggestions and criticisms.

Amino Acid Sequence↗
PubMed1998

Quality assessment of NMR structures: a statistical survey.

A statistical analysis is reported of experimental data and coordinates of a set of 97 NMR structures deposited in the PDB. The aim is to assess the quality of these structures in relation to the amount of experimental information. Experimental restraints were analysed using the program AQUA. Many nomenclature inconsistencies between deposited restraint and coordinate files were observed. The experimental restraint files were found to contain a high proportion of redundant restraints. Procedures for analysing and correcting the inconsistencies and restraint counts are described. The analysis of NOE restraint violations (using AQUA) and of a wide variety of geometrical quality indicators (using PROCHECK-NMR and WHAT IF) provides a reference for other NMR structure determinations. The extent of NOE violations is anti-correlated with the quality of the Ramachandran map. The precision as measured by the circular variance of backbone dihedral angles, does increase with the amount of experimental data, as expected, but is sometimes overestimated. Bond lengths, bond angles and planarity of groups can deviate considerably from ideal values. Outliers appear to cluster per laboratory, indicating that the results depend on particulars of refinement protocols and/or software. We have identified a problem of atom overlap in a number of refined structures.We recommend adhering to the standard nomenclature as put forward by an IUPAC Task Group, to ensure consistency between restraints and coordinates, and to omit redundant restraints from the deposition. The results obtained from this analysis and the AQUA program are available through the World Wide Web.

Databases, Factual↗
PubMed1998

Recommendations for the presentation of NMR structures of proteins and nucleic acids.

The recommendations presented here are designed to support easier communication of NMR data and NMR structures of proteins and nucleic acids through unified nomenclature and reporting standards. Much of this document pertains to the reporting of data in journal articles; however, in the interest of the future development of structural biology, it is desirable that the bulk of the reported information be stored in computer-accessible form and be freely accessible to the scientific community in standardized formats for data exchange. These recommendations stem from an IUPAC-IUBMB-IUPAB inter-union venture with the direct involvement of ICSU and CODATA. The Task Group has reviewed previous formal recommendations and has extended them in the light of more recent developments in the field of biomolecular NMR spectroscopy. Drafts of the recommendations presented here have been examined critically by more than 50 specialists in the field and have gone through two rounds of extensive modification to incorporate suggestions and criticisms.

Databases as Topic↗
PubMed1998

Mobilities of the inner three core residues and the Man(alpha 1--6) branch of the glycan at Asn78 of the alpha-subunit of human chorionic gonadotropin are restricted by the protein.

Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone involved in the maintenance of the corpus luteum in early pregnancy. The free alpha-subunit of hCG has a biological activity of its own, namely, stimulation of prolactin secretion from term pregnancy decidual cells [Blithe, D. L., et al. (1991) Endocrinology 129, 2257-2259]. Glycosylation at Asn78 of the alpha-subunit is required for the stability of the protein, but the exact nature of the stabilizing effect is not known. In our previous study, it was indicated that GlcNAc-1 at Asn78 has a reduced mobility, whereas the glycan at Asn52 is highly mobile [De Beer, T., et al. (1996) Eur. J. Biochem. 241, 229-242]. In the present investigation, it is shown that the PNGase F susceptibility of the Asn52-linked glycan in the free alpha-subunit is absent in the heterodimer. Thus, the high mobility of the glycan at Asn52 may be characteristic for the free alpha-subunit. For accurate modeling of alpha hCG, knowledge of the behavior of each of the glycans is essential. In this context, the mobility of the glycans and their interactions with the protein are explored by NMR spectroscopy using desialylated, partially deglycosylated free alpha-subunit (as-pd alpha) carrying glycans at Asn78 only. NOEs between GlcNAc-2 and several amino acid residues indicate that GlcNAc-2 is involved in stabilizing alpha hCG. From the values of 13C relaxation parameters T2 and T1 rho of the constituting monosaccharide residues, it was concluded that the inner three residues have a severely restricted mobility. The Man-4 and Man-4' residues of the diantennary oligosaccharide exhibit a similar relaxation behavior, suggesting that the Man-4' branch occurs in a single conformation of the C5-C6 linkage of Man-3 instead of in rapidly interconverting conformations that are known to exist for this linkage for the free oligosaccharide.

Asparagine↗
PubMed1998

An Off-resonance Rotating Frame Relaxation Experiment for the Investigation of Macromolecular Dynamics Using Adiabatic Rotations

15N off-resonance rotating frame relaxation can be applied to the study of internal dynamics in proteins in the millisecond to microsecond regime. We show that the performance of existing methods can be improved by application of simultaneous amplitude and phase-modulated adiabatic RF pulses to align the nuclear spin magnetization with the off-resonance spin-lock field for all the spins under investigation. Application of this technique to the 269-residue serine protease PB92 allowed the measurement of 15N off-resonance rotating frame relaxation rates for all nonoverlapping residues in the protein, including the arginine side chains, encompassing a chemical shift range of 50 ppm. Simulations indicate that by use of the proposed adiabatic RF pulses rotating frame relaxation rates can be obtained for magnetization vectors aligned at arbitrary angles with the static field. Copyright 1998 Academic Press.

Journal Article↗
PubMed1998

Recommendations for the presentation of NMR structures of proteins and nucleic acids. IUPAC-IUBMB-IUPAB Inter-Union Task Group on the Standardization of Data Bases of Protein and Nucleic Acid Structures Determined by NMR Spectroscopy.

The recommendations presented here are designed to support easier communication of NMR data and NMR structures of proteins and nucleic acids through unified nomenclature and reporting standards. Much of this document pertains to the reporting of data in journal articles; however, in the interest of the future development of structural biology, it is desirable that the bulk of the reported information be stored in computer-accessible form and be freely accessible to the scientific community in standardized formats for data exchange. These recommendations stem from an IUPAC-IUBMB-IUPAB inter-union venture with the direct involvement of ICSU and CODATA. The Task Group has reviewed previous formal recommendations and has extended them in the light of more recent developments in the field of biomolecular NMR spectroscopy. Drafts of the recommendations presented here have been examined critically by more than 50 specialists in the field and have gone through two rounds of extensive modification to incorporate suggestions and criticisms.

Amino Acid Sequence↗
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

The solution structure of the amino-terminal HHCC domain of HIV-2 integrase: a three-helix bundle stabilized by zinc.

BACKGROUND: Integrase mediates a crucial step in the life cycle of the human immunodeficiency virus (HIV). The enzyme cleaves the viral DNA ends in a sequence-dependent manner and couples the newly generated hydroxyl groups to phosphates in the target DNA. Three domains have been identified in HIV integrase: an amino-terminal domain, a central catalytic core and a carboxy-terminal DNA-binding domain. The amino-terminal region is the only domain with unknown structure thus far. This domain, which is known to bind zinc, contains a HHCC motif that is conserved in retroviral integrases. Although the exact function of this domain is unknown, it is required for cleavage and integration. RESULTS: The three-dimensional structure of the amino-terminal domain of HIV-2 integrase has been determined using two-dimensional and three-dimensional nuclear magnetic resonance data. We obtained 20 final structures, calculated using 693 nuclear Overhauser effects, which display a backbone root-mean square deviation versus the average of 0.25 A for the well defined region. The structure consists of three alpha helices and a helical turn. The zinc is coordinated with His 12 via the N epsilon 2 atom, with His16 via the N delta 1 atom and with the sulfur atoms of Cys40 and Cys43. The alpha helices form a three-helix bundle that is stabilized by this zinc-binding unit. The helical arrangement is similar to that found in the DNA-binding domains of the trp repressor, the prd paired domain and Tc3A transposase. CONCLUSION: The amino-terminal domain of HIV-2 integrase has a remarkable hybrid structure combining features of a three-helix bundle fold with a zinc-binding HHCC motif. This structure shows no similarity with any of the known zinc-finger structures. The strictly conserved residues of the HHCC motif of retroviral integrases are involved in metal coordination, whereas many other well conserved hydrophobic residues are part of the protein core.

Amino Acid Sequence↗
PubMed1997

Involvement of laser photo-CIDNP (chemically induced dynamic nuclear polarization)-reactive amino acid side chains in ligand binding by galactoside-specific lectins in solution.

For proteins in solution the validity of certain crystallographic parameters can be ascertained by a combination of molecular-dynamics (MD) simulations and NMR spectroscopy. Using the laser photo-CIDNP (chemically induced dynamic nuclear polarization) technique as a measure for surface accessibility of histidine, tyrosine and tryptophan, the spectra of bovine galectin-1 and Erythrina corallodendron lectin (EcorL) are readily reconcilable with the crystallographic data for these two proteins. The results emphasise the role of Trp68/Trp69 for carbohydrate binding in bovine galectin-1/chicken galectins and of Trp194 in murine galectin-3. This feature derived from the crystal structure of bovine galectin-1 is maintained in solution for the prototype human homologue, two avian galectins and the chimera-type murine galectin-3, as the spectra corroborate the CIDNP-inferable spatial parameters of the four calculated models for binding-site architecture. In EcorL, Tyr106/Tyr108 are constituents of the extended combining pocket, which can be shielded in solution by ligand presence. Discrepancies between results from modelling and CIDNP measurements concern primarily the lack of reactivity of histidine residues for human and avian prototype galectins and of Tyr82/Tyr229 of the plant lectin. Site-directed mutagenesis of EcorL is assumed to provide information on the role of a certain residue for functional aspects. When single-site mutants of EcorL ([Ala106]EcorL, [Ala108]EcorL, [Ala229]EcorL) were subjected to molecular-dynamics (MD) simulations, the apparent surface accessibilities even of spatially separated amino acid side chains could non-uniformly be affected. This conclusion is supported by the assessment of the spectra for the mutant proteins. On the basis of these CIDNP-results modelling of the binding-site architecture of the lectin indicates the occurrence of notable alterations in the orientation of Tyr106/Tyr108 phenyl rings. The implied potential effect of single-site mutations on conformational features of a protein will deserve attention for the interpretation of studies comparing wild-type and mutant proteins.

Amino Acid Sequence↗
PubMed1997

The structure of the translational initiation factor IF1 from E.coli contains an oligomer-binding motif.

The structure of the translational initiation factor IF1 from Escherichia coli has been determined with multidimensional NMR spectroscopy. Using 1041 distance and 78 dihedral constraints, 40 distance geometry structures were calculated, which were refined by restrained molecular dynamics. From this set, 19 structures were selected, having low constraint energy and few constraint violations. The ensemble of 19 structures displays a root-mean-square deviation versus the average of 0.49 A for the backbone atoms and 1.12 A for all atoms for residues 6-36 and 46-67. The structure of IF1 is characterized by a five-stranded beta-barrel. The loop connecting strands three and four contains a short 3(10) helix but this region shows considerably higher flexibility than the beta-barrel. The fold of IF1 is very similar to that found in the bacterial cold shock proteins CspA and CspB, the N-terminal domain of aspartyl-tRNA synthetase and the staphylococcal nuclease, and can be identified as the oligomer-binding motif. Several proteins of this family are nucleic acid-binding proteins. This suggests that IF1 plays its role in the initiation of protein synthesis by nucleic acid interactions. Specific changes of NMR signals of IF1 upon titration with 30S ribosomal subunit identifies several residues that are involved in the interaction with ribosomes.

Amino Acid Sequence↗