Biomedical subjects

R Boelens

Publications and source records attributed to R Boelens.

At least 109 records · Page 6Linked to original sources

PubMed1991

Structure determination of a DNA octamer in solution by NMR spectroscopy. Effect of fast local motions.

NMR structures of biomolecules are primarily based on nuclear Overhauser effects (NOEs) between protons. For the interpretation of NOEs in terms of distances, usually the assumption of a single rotational correlation time corresponding to a rigid molecule approximation is made. Here we investigate the effect of fast internal motions of the interproton vectors in the context of the relaxation matrix approach for structure determination of biomolecules. From molecular dynamics simulations generalized order parameters were calculated for the DNA octamer d(GCGTTCGC).d(CGCAACGC), and these were used in the calculation of NOE intensities. The magnitudes of the order parameters showed some variation for the different types of interproton vectors. The lowest values were observed for the interresidue base H6/H8-H2" proton vectors (S2 = 0.60), while the cytosine H5-H6 interproton vectors were among the most motionally restricted (S2 = 0.92). Inclusion of the motion of the interproton vectors resulted in a much better agreement between theoretically calculated NOE spectra and the experimental spectra measured by 2D NOE spectroscopy. The interproton distances changed only slightly, with a maximum of 10%; nevertheless, the changes were significant and resulted in constraints that were better satisfied. The structure of the DNA octamer was determined by using restrained molecular dynamics simulations with H2O as a solvent, with and without the inclusion of local internal motions. Starting from A- or B-DNA, the structures showed a high local convergence (0.86 A), while the global convergence for the octamer was ca. 2.6 A.

Base Sequence↗

PubMed1991

Porcine pancreatic phospholipase A2: sequence-specific 1H and 15N NMR assignments and secondary structure.

The solution structure of porcine pancreatic phospholipase A2 (124 residues, 14 kDa) has been studied by two-dimensional homonuclear 1H and two- and three-dimensional heteronuclear 15N-1H nuclear magnetic resonance spectroscopy. Backbone assignments were made for 117 of the 124 amino acids. Short-range nuclear Overhauser effect (NOE) data show three alpha-helices from residues 1-13, 40-58, and 90-109, an antiparallel beta-sheet for residues 74-85, and a small antiparallel beta-sheet between residues 25-26 and 115-116. A 15N-1H heteronuclear multiple-quantum correlation experiment was used to monitor amide proton exchange over a period of 22 h. In total, 61 amide protons showed slow or intermediate exchange, 46 of which are located in the three large helices. Helix 90-109 was found to be considerably more stable than the other helices. For the beta-sheets, four hydrogen bonds could be identified. The secondary structure of porcine PLA in solution, as deduced from NMR, is basically the same as the structure of porcine PLA in the crystalline state. Differences were found in the following regions, however. Residues 1-6 in the first alpha-helix are less structured in solution than in the crystal structure. Whereas in the crystal structure residues 24-29 are involved both in a beta-sheet with residues 115-117 and in a hairpin turn, the expected hydrogen bonds between residues 24-117 and 25-29 do not show slow exchange behavior. This and the absence of several expected NOEs imply that this region has a less well defined structure in solution. Finally, the hydrogen bond between residues 78-81, which is part of a beta-sheet, does not show slow exchange behavior.

Amino Acid Sequence↗

PubMed1991

Computer-assisted assignment of 2D 1H NMR spectra of proteins: basic algorithms and application to phoratoxin B.

A suite of computer programs (CLAIRE) is described which can be of assistance in the process of assigning 2D 1H NMR spectra of proteins. The programs embody a software implementation of the sequential assignment approach first developed by Wüthrich and co-workers (K. Wüthrich, G. Wider, G. Wagner and W. Braun (1982) J. Mol. Biol. 155, 311). After data-abstraction (peakpicking), the software can be used to detect patterns (spin systems), to find cross peaks between patterns in 2D NOE data sets and to generate assignments that are consistent with all available data and which satisfy a number of constraints imposed by the user. An interactive graphics program called CONPAT is used to control the entire assignment process as well as to provide the essential feedback from the experimental NMR spectra. The algorithms are described in detail and the approach is demonstrated on a set of spectra from the mistletoe protein phoratoxin B, a homolog of crambin. The results obtained compare well with those reported earlier based entirely on a manual assignment process.

Algorithms↗

PubMed1991

Photo-CIDNP study of the interaction between the glucocorticoid receptor DNA-binding domain and glucocorticoid response elements.

Photo-CIDNP studies were performed on two protein fragments that both contain the double zinc-finger DNA-binding domain of the glucocorticoid receptor. In the absence of DNA, Tyr452 and Tyr474 are polarised in both fragments while Tyr497 is not. Addition of a palindromic glucocorticoid response element (GRE) results in the suppression of Tyr474 polarization while the polarization of Tyr452 is unaffected. The same result is observed upon adding a half GRE to the protein fragment indicating that the suppression of Tyr474 polarization is not due to protein-protein contacts but to interaction with DNA.

Amino Acid Sequence↗

PubMed1991

Direct NOE refinement of biomolecular structures using 2D NMR data.

A set of computer programs called DINOSAUR has been developed, which allows the refinement of biomolecular structures directly from 2D NOE intensities. The NOE restraining potential implemented emphasises the weak intensities corresponding to larger distances which are more likely to determine the three-dimensional structure. An approximation based on a two-spin approximation is proposed for the gradient of the NOE intensities instead of the exact solution which is extremely time-consuming. The DINOSAUR routines have been implemented in various refinement programs (Distance bound Driven Dynamics, Molecular Dynamics and Energy Minimisation) and tested on an eight-residue model peptide.

Magnetic Resonance Spectroscopy↗

PubMed1991

Statistical analysis of double NOE transfer pathways in proteins as measured in 3D NOE-NOE spectroscopy.

The recent development of three-dimensional NMR spectroscopy has alleviated the problem of overlap of resonances. However, also for the 3D experiments resonance assignment strategies have usually relied upon knowledge about spin systems, combined with information about short (sequential) distances. For doubly (15N/13C)-labelled molecules, a novel assignment strategy has been developed. In this paper we address the possibilities of an assignment strategy for proteins, based solely upon the use of NOE data. For this, the 3D NOE-NOE experiment seems most suitable. Therefore, we have made a theoretical evaluation of double NOE transfer pathways in 28 protein crystal structures. We identify 95 connectivities which are most likely to be observed as cross peaks in a 3D NOE-NOE spectrum of a protein. Given the occurrence of one of these 95 connectivities, we evaluate the chances of occurrence for the others. Analysis of these conditional probabilities allowed the construction of five patterns of related, highly correlated cross peaks which resemble the conventional idea of spin systems to some extent and may provide a basis for assignment and secondary structure analysis from 3D NOE-NOE data alone.

Enzymes↗

PubMed1990

Assignment of the 1H-NMR spectrum of a lac repressor headpiece-operator complex in H2O and identification of NOEs. Consequences for protein-DNA interaction.

A complex between the headpiece amino-terminal residues 1-56 of lac repressor (HP56) and an 11-bp lac operator fragment was studied by 1H NMR. The sequence specific assignment of the exchangeable and non-exchangeable protons has been accomplished. Several protons have favourable chemical shifts in the complex, therefore new intraprotein NOEs could be found that had not been unambigously identified in the free protein. By comparison, most of these intraprotein NOEs are also present in the spectra of the free headpiece but some are different. Furthermore, several new proteins DNA NOEs could be identified. The NOE between the side-chain amide protons of Gln18 and C5H of C7 confirms the specific contact between these residues which was proposed from genetic experiments [Ebright, R. M. (1985) J. Biomol. Struct. & Dyn. 3, 281-297]. The implications of the new data for the interaction between the lac repressor headpiece and its operator are discussed.

DNA-Binding Proteins↗

PubMed1990

1H NMR studies of the glucocorticoid receptor DNA-binding domain: sequential assignments and identification of secondary structure elements.

Two protein fragments containing the DNA-binding domain (DBD) of the glucocorticoid receptor (GR) have been studied by two-dimensional 1H NMR spectroscopy. The two peptides (93 and 115 residues, respectively) contain a common segment corresponding to residues C440-I519 of the rat GR or residues C421-I500 of the human GR and include two Zn-binding "finger" domains. The structures of this segment are almost identical in the two protein fragments, as judged from chemical shifts and sequential NOE connectivities. More than 90% of all observable 1H resonances within a 71-residue segment encompassing C440-R510 (rat GR) could be sequentially assigned by standard techniques, and stereospecific assignments could be made for the methyl groups in four valine residues within this segment. Sequential NOE connectivities indicate several elements of secondary structure including two alpha-helical segments consisting of residues S459-E469 and P493-G504, a type I reverse turn between residues R479 and C482, a type II reverse turn between residues L475 and G478, and several regions of extended peptide conformation. No evidence for alpha-helical conformation was found within the two putative zinc-finger domains, indicating that the structures of these domains differ from that of TFIIIA-type zinc fingers. The observation of some very slowly exchanging amide protons in the N-terminal (CI) domain of the DBD in combination with slow rotation of the Y452 aromatic ring indicates that this domain has a restricted conformational flexibility compared to the C-terminal (CII) domain. We also observe several long-range NOE connectivities within C440-R510, suggesting that the sequential assignments presented here will provide a basis for a complete structure determination of this segment of the GR.

Amino Acid Sequence↗

PubMed1990

Structure of Arc repressor in solution: evidence for a family of beta-sheet DNA-binding proteins.

The Arc repressor, which is involved in the switch between lysis and lysogeny of Salmonella bacteriophage P22, does not belong to any of the known classes of DNA-binding proteins. Mutagenesis studies show that the DNA-binding region is located in the 15 N-terminal amino-acid residues. We have now determined the three-dimensional structure of the Arc dimer from an extensive set of interproton-distance data obtained from 1H NMR spectroscopy. A priori, intra- and inter-monomer nuclear Overhauser effects (NOEs) cannot be distinguished for a symmetric dimer. But by using the homology with the Escherichia coli Met repressor we could interpret the NOEs unambiguously in an iterative structure refinement procedure. The final structure satisfies a large set of NOE constraints (1,352 for the dimer). It shows a strongly intertwined dimer, in which residues 8-14 of different monomers form an antiparallel beta-sheet. A model for the Arc repressor-operator complex can account for all available biochemical and genetic data. In this model two Arc dimers bind with their beta-sheet regions in successive major grooves on one side of the DNA helix, similar to the Met repressor interaction. Thus, Arc and Met repressors are members of the same family of proteins, which contain an antiparallel beta-sheet as the DNA-binding motif.

Amino Acid Sequence↗

PubMed1990

Two-dimensional NMR studies on des-pentapeptide-insulin. Proton resonance assignments and secondary structure analysis.

The shortened analogue of insulin, des-(B26-B30)-pentapeptide insulin, has been characterized by two-dimensional 1H NMR. The 1H resonance assignments and the secondary structure in water solution are discussed The results indicate that the secondary structure in solution is very similar to that reported for the crystalline state. A high flexibility of both A and B chains is observed. Of the two conformations seen in the 2-Zn insulin crystals and indicated as molecules 1 and 2 (Chinese nomenclature), the structure of the analogue is more similar to that of molecule 1.

Amino Acid Sequence↗

PubMed1990

Solution structure of the glucocorticoid receptor DNA-binding domain.

The three-dimensional structure of the DNA-binding domain (DBD) of the glucocorticoid receptor has been determined by nuclear magnetic resonance spectroscopy and distance geometry. The structure of a 71-residue protein fragment containing two "zinc finger" domains is based on a large set of proton-proton distances derived from nuclear Overhauser enhancement spectra, hydrogen bonds in previously identified secondary structure elements, and coordination of two zinc atoms by conserved cysteine residues. The DBD is found to consist of a globular body from which the finger regions extend. A model of the dimeric complex between the DBD and the glucocorticoid response element is proposed. The model is consistent with previous results indicating that specific amino acid residues of the DBD are involved in protein-DNA and protein-protein interactions.

Amino Acid Sequence↗

PubMed1990

Two-dimensional NMR study of a protein-DNA complex. lac repressor headpiece-operator interaction.

The interaction of the N-terminal DNA-binding domain (56 amino acid residues) of the lac repressor with lac operator DNA was analyzed using two-dimensional NMR spectroscopy. Both half-operators (11 and 14 bp) and a complete fully symmetric 22 bp operator were studied. Two-dimensional nuclear Overhauser effect (2D NOE) spectra of headpiece-operator complexes were taken in both D2O and H2O solutions. Special attention was given to the problem of 1H resonance assignments. Based on an analysis of the proton-proton NOEs, a model for the headpiece-operator complex could be derived. In this model, most of the protein-DNA contracts occur between amino acid residues in the second helix (recognition helix) of the lac headpiece and DNA bases in the major groove. The orientation of this helix with respect to the dyad axis of the operator is opposite to that found in the X-ray structures of several other repressor-operator complexes.

Base Composition↗

PubMed1990

Assignment strategies in homonuclear three-dimensional 1H NMR spectra of proteins.

The increase in dimensionality of three-dimensional (3D) NMR greatly enhances the spectral resolution in comparison to 2D NMR. It alleviates the problem of resonance overlap and may extend the range of molecules amenable to structure determination by high-resolution NRM spectroscopy. Here, we present strategies for the assignment of protein resonances from homonuclear nonselective 3D NOE-HOHAHA spectra. A notation for connectivities between protons, corresponding to cross peaks in 3D spectra, is introduced. We show how spin systems can be identified by tracing cross-peak patterns in cross sections perpendicular to the three frequency axes. The observable 3D sequential connectivities in proteins are tabulated, and estimates for the relative intensities of the corresponding cross peaks are given for alpha-helical and beta-sheet conformations. Intensities of the cross peaks in the 3D spectrum of pike III parvalbumin follow the predictions. The sequential-assignment procedure is illustrated for loop regions, extended and alpha-helical conformations for the residues Ala 54-Leu 63 of parvalbumin. NOEs that were not previously identified in 2D spectra of parvalbumin due to overlap are found.

Hydrogen↗

PubMed1989

Sequence-specific 1H NMR assignment and secondary structure of the Arc repressor of bacteriophage P22, as determined by two-dimensional 1H NMR spectroscopy.

The Arc repressor of bacteriophage P22 is a DNA binding protein that does not belong to any of the known classes of such proteins. We have undertaken a 1H NMR study of the protein with the aim of elucidating its three-dimensional structure in solution and its mode of binding of operator DNA. Here we present the 1H nuclear magnetic resonance (NMR) assignments of all backbone protons and most of the side-chain protons of Arc repressor. Elements of secondary structure have been identified on the basis of networks of characteristic sequential and medium-range nuclear Overhauser enhancements (NOEs). Two alpha-helical regions have been found in the peptide regions 16-29 and 35-45. The ends of the helices could not yet be firmly established and could extend to residue 31 for the first helix and to residue 49 for the second. Immediately before the first helix, between residues 8 and 14, a region is present with beta-sheet characteristics dominated by a close proximity of the alpha-protons of residues 9 and 13. Because of the dimeric nature of the protein there are still two possible ways in which the NOEs in the beta-sheet region can be interpreted. If the NOEs are intramonomer, this requires a tight turn involving residues 10-12. Alternatively, if the NOEs are intermonomer, then and antiparallel beta-sheet would be implicated comprising two strands of different Arc monomers. While the data presently do not allow an unambiguous choice between these two possibilities, some evidence is discussed that favors the latter (beta-sheet between monomers).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1989

1H NMR studies of bovine and porcine phospholipase A2: assignment of aromatic resonances and evidence for a conformational equilibrium in solution.

Bovine and porcine pancreatic phospholipases A2, and porcine isophospholipase A2, have been investigated by one- and two-dimensional 1H NMR spectroscopy. Resonances have been assigned for 20-26 residues in each enzyme, including all the aromatic residues, by a strategy based on the semiquantitative comparison of proximity relationships deduced from NOE experiments with those seen in the crystal structure NOE experiments indicate that the loop comprising residues 59-70, which has a different conformation in the crystal structures of the bovine and porcine enzymes, has the same conformation in these two enzymes in solution. Selective changes in the line width of a limited number of resonances as a function of pH, temperature, and calcium concentration provide evidence for a local conformational equilibrium. This equilibrium involves a limited region of the protein structure around residues 25, 41, 106, and 111; it has been identified in the bovine enzyme and porcine isoenzyme but is not apparent in the porcine enzyme.

Animals↗

PubMed1989

H NMR study of a complex between the lac repressor headpiece and a 22 base pair symmetric lac operator.

A complex between the lac repressor headpiece and a fully symmetric tight-binding 22 bp lac operator was studied by 2D NMR. Several 2D NOE spectra were recorded for the complex in both H2O and 2H2O. Many NOE cross-peaks between the headpiece and DNA could be identified, and changes in the chemical shift of the DNA protons upon complex formation were analyzed. Comparison of these data with those obtained for a complex between the headpiece and a 14 bp half-operator, studied previously [Boelens, R., Scheek, R. M., Lamerichs, R. M. J. N., de Vlieg, J., van Boom, J. H., & Kaptein, R. (1987) in DNA-ligand interactions (Guschlbauer, W., & Saenger, W., Eds.) pp 191-215, Plenum, New York], shows that two headpieces form a specific complex with the 22 bp lac operator in which each headpiece binds in the same way as found for the 14 bp complex. The orientation of the recognition helix in the major groove of DNA in these complexes is opposite with respect to the dyad axis to that found for other repressors.

Base Composition↗

PubMed1989

Ligand-binding effects on the kringle 4 domain from human plasminogen: a study by laser photo-CIDNP 1H-NMR spectroscopy.

Photo-chemically induced dynamic nuclear polarization (photo-CIDNP) one-dimensional and two-dimensional (2D) 1H-NMR techniques have been applied to the study of the kringle 4 domain of human plasminogen both ligand-free and complexed to the antifibrinolytic drugs epsilon-aminocaproic acid and p-benzylaminesulfonic acid (BASA). A number of aromatic side-chains (His3, Trp72, Tyr41, Tyr50 and Tyr74) appear to be exposed and accessible to 3-N-carboxymethyl-lumiflavin, the photopolarizing flavin dye, both in the presence and in the absence of ligands. A lesser exposure is observed for the Trp25 and Trp62 indole groups in the presence of BASA. The spin-spin (J-coupling) and dipolar (Overhauser) connectivities in the 2D experiments afford absolute assignment of aromatic resonances for the above residues, as well as of those stemming from the Trp72 ring in the presence of BASA. Moreover, a number of H beta resonances can be identified and sorted according to specific types of amino acid residues.

Aminocaproic Acid↗

PubMed1989

Sequence-specific 1H-n.m.r. assignments and peptide backbone conformation in rat epidermal growth factor.

The solution conformation of rat epidermal growth factor (EGF) has been investigated by proton n.m.r. techniques. Two-dimensional proton n.m.r. experiments have allowed sequential resonance assignments to be made for most protons. On the basis of these assignments, two regions of anti-parallel beta-sheet structure have been derived from the n.m.r. data. A beta-sheet segment running from about V19 to V23 (capital letters refer to amino acids in the single-letter notation) is folded onto a beta-sheet segment running from R28 to N32 and joined by a chain reversal from E24 to D27. A second region involves a beta-turn from V34 to Y37, which starts a short beta-sheet up to G39, followed by a chain reversal up to Q43, which leads to folding of the C-terminal beta-sheet segment, i.e. H44-R45, running antiparallel to the short Y37 beta-sheet segment. The N-terminal segment up to G18 exists in a multiple bend conformation and is folded on to the V29-V23/R28-N32 beta-sheet such that Y10, Y13, Y22 and Y29 are proximal to each other. Structural comparison of rat, murine and human EGFs indicates a number of highly conserved structural features common to at least these species of EGF.

Animals↗