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Biomedical subjects

R Boelens

Publications and source records attributed to R Boelens.

At least 91 records · Page 5Linked to original sources
PubMed1993

Structure of the complex of lac repressor headpiece and an 11 base-pair half-operator determined by nuclear magnetic resonance spectroscopy and restrained molecular dynamics.

The structure of the complex of lac repressor headpiece and an 11 base-pair lac half-operator has been determined by NMR spectroscopy and restrained Molecular Dynamics calculations. In total 508 distances were derived from two-dimensional nuclear Overhauser enhancement measurements, 260 of which are within the headpiece, 212 within the operator and 36 between operator and headpiece. An equilibrium restrained Molecular Dynamics calculation of the complex in aqueous solution, spanning 85 picoseconds, has been used to analyze the structure. Configuration sampling by an annealing procedure has been undertaken as well in order to estimate the precision of the structure determination. Our data confirm the results of previous two-dimensional NMR studies that the orientation of the recognition helix of lac repressor in the major groove of DNA with respect to the operator dyad axis is opposite to the orientation found in complexes of other DNA binding proteins of the helix-turn-helix class. We find a number of tight contacts between the protein and the operator that are in agreement with the available genetic and biochemical data. The anchoring of lac headpiece on the operator is similar to that of other repressors. Other features are unique for lac headpiece: relative few direct hydrogen bonds between side-chains and bases; extensive apolar contacts; many direct and water-bridged contacts to phosphates from residues in or close to the recognition helix. Overall, an interconnected set of interactions is observed, involving base-specific contacts, phosphate contacts, intra-protein and water-bridged hydrogen bonds. Several of these interactions appear to be dynamic, i.e. fluctuating in time, rather than static.

Amino Acid Sequence↗
PubMed1993

Observation of inter-subunit nuclear Overhauser effects in a dimeric protein. Application to the Arc repressor.

For the structure determination of symmetric protein dimers it is necessary to distinguish between intra- and inter-subunit NOEs. A method is presented to measure selectively the inter-subunit NOEs using uniform 15N and 13C isotope labelling. This is accomplished by double filtered 2D NOE experiments on mixtures of native protein with isotope-labeled protein. The method has been applied to the Arc repressor and allows the characterization of virtually all proton-proton NOEs in terms of their intra- or inter-subunit nature.

Bacterial Proteins↗
PubMed1993

NMR studies of the POU-specific DNA-binding domain of Oct-1: sequential 1H and 15N assignments and secondary structure.

The 1H and 15N resonances of the POU-specific DNA-binding domain of transcription factor Oct-1 have been assigned sequentially using two-dimensional homo- and heteronuclear NMR techniques, as well as three-dimensional heteronuclear NMR techniques, including TOCSY, 2D NOE, and NOESY-HMQC experiments. A number of typical short- and medium-range NOE contacts, as well as amide proton exchange data, gave evidence for the presence of four alpha-helices, in the peptide segments 1-19, 23-34, 40-49, and 54-71, which are connected by short loops of irregular structure. Interestingly, the second helix contains three glycine residues and the fourth helix a proline in the middle of the helix. Although the regular pattern of hydrogen bonds in the fourth helix is interrupted, due to the absence of an amide proton in proline, the helix is remarkably stable. All four helices are amphipathic, which suggests a packing of the apolar sides of the helices in the folded structure of the protein.

Amino Acid Sequence↗
PubMed1993

Heteronuclear 113Cd-1H NMR study of metal coordination in the human retinoic acid receptor-beta DNA binding domain.

The two zinc fingers of the DNA binding domain of the human retinoic acid receptor-beta were labelled with 113Cd. Two- and three-dimensional heteronuclear nuclear magnetic resonance (NMR) experiments show that the first eight conserved cysteine residues coordinate the two zinc ions tetrahedrally. The ninth conserved cysteine is not involved in metal coordination. In each finger one cysteine exhibits a heteronuclear 113Cd-1H coupling constant substantially smaller than those of the other metal binding cysteines.

Amino Acid Sequence↗
PubMed1993

Solution structure of the POU-specific DNA-binding domain of Oct-1.

The transcription factor Oct-1 belongs to a family containing a POU DNA-binding domain. This bipartite domain is composed of a POU-specific domain (POUs) and a POU-homeodomain (POUhd) connected by a flexible linker. The left half of the optimal POU binding site, the octamer ATGCAAAT, is recognized by POUs and the right half by POUhd. We have determined the solution structure of POUs by nuclear magnetic resonance. It consists of four alpha-helices connected by short loops. Helices I and IV are in a parallel coiled-coil arrangement. The folding topology appears to be similar to that of the bacteriophage lambda-repressor and 434 repressor. For the well defined parts of the protein (residues 1-71), the average root-mean square deviation for the backbone atoms is 0.9 A. Based on the observed selective exchange broadening in the (15N,1H)-HMQC (heteronuclear multiple quantum coherence) spectrum of the POUs-DNA complex we conclude that DNA-binding is mediated by helix III. We propose a model for the POU-DNA complex in which both recognition helices from the two subdomains have adjacent positions in the major groove.

Amino Acid Sequence↗
PubMed1993

"Ensemble" iterative relaxation matrix approach: a new NMR refinement protocol applied to the solution structure of crambin.

The structure in solution of crambin, a small protein of 46 residues, has been determined from 2D NMR data using an iterative relaxation matrix approach (IRMA) together with distance geometry, distance bound driven dynamics, molecular dynamics, and energy minimization. A new protocol based on an "ensemble" approach is proposed and compared to the more standard initial rate analysis approach and a "single structure" relaxation matrix approach. The effects of fast local motions are included and R-factor calculations are performed on NOE build-ups to describe the quality of agreement between theory and experiment. A new method for stereospecific assignment of prochiral groups, based on a comparison of theoretical and experimental NOE intensities, has been applied. The solution structure of crambin could be determined with a precision (rmsd from the average structure) of 0.7 A on backbone atoms and 1.1 A on all heavy atoms and is largely similar to the crystal structure with a small difference observed in the position of the side chain of Tyr-29 which is determined in solution by both J-coupling and NOE data. Regions of higher structural variability (suggesting higher mobility) are found in the solution structure, in particular for the loop between the two helices (Gly-20 to Pro-22).

Amino Acid Sequence↗
PubMed1993

The solution structure of the human retinoic acid receptor-beta DNA-binding domain.

The three-dimensional structure of the DNA-binding domain of the human retinoic acid receptor-beta (hRAR-beta) has been determined by nuclear magnetic resonance spectroscopy in conjunction with distance geometry, restrained molecular dynamics and iterative relaxation matrix calculations. A total of 1244 distance restraints were obtained from NOE intensities, of which 448 were intra-residue and 796 inter-residue restraints. In addition 23 chi and 30 phi dihedral angle restraints were obtained from J-coupling data. The two 'zinc-finger' regions of the 80-amino acid residue protein are followed by two alpha-helices that cross each other perpendicularly. There is a short stretch of b-sheet near the N-terminus. The alpha-helical core of the protein is well determined with a backbone root-mean-square deviation (r.m.s.d.) with respect to the average of 0.18 A and 0.37 A when the side chains of residues 31, 32, 36, 61, 62, 65 and 69 are included. The r.m.s.d. for the backbone of residues 5-80 is 0.76 A. For the first finger (residues 8-28), the r.m.s.d. of the backbone is 0.79 A. For the second finger (residues 44-62) the r.m.s.d. is 0.64 A. The overall structure is similar to that of the corresponding domain of the glucocorticoid receptor, although the C-terminal part of the protein is different. The second alpha-helix is two residues shorter and is followed by a well-defined region of extended backbone structure.

Amino Acid Sequence↗
PubMed1992

Conformational changes in phospholipase A2 upon binding to micellar interfaces in the absence and presence of competitive inhibitors. A 1H and 15N NMR study.

An NMR study has been made of porcine pancreatic phospholipase A2 (PLA) in three environments: free in solution, in a binary complex with dodecylphosphocholine micelles, and in a ternary complex with a micelle and the substrate-like inhibitor (R)-1-octyl-2-(N-dodecanoylamino)-2-deoxyglycero-3-phosph oglycol. 1H and 15N chemical shifts, amide exchange rates, and NOE intensities are compared for the enzyme in different environments. From these data, structural differences are found for the N-terminal part, the end of the surface loop at residues Tyr69 and Thr70, and the active site residue His48, and also for the Ca-binding loop (residues 28-32). Specifically, when binding to a micelle, the side chains of residues Ala1, Trp3, and Tyr69, as well as all protons of Thr70, are found to be closer together. After subsequent introduction of the competitive inhibitor, further changes are found for these residues. The N-terminus is flexible in PLA free in solution, in contrast with the crystal structures where it adopts an alpha-helical conformation. According to the NMR data, this helix is rigidly formed only in the ternary complex. Furthermore, in the ternary complex, the N-terminal amino group and the exchangeable hydrogen at N3 of the ring of His48 are observed. We propose that PLA is activated in two steps. An initial conformational change occurs upon binding to a micellar interface. The catalytically active conformation of the enzyme, which has an extensive network of hydrogen bonds, is formed only when binding a substrate or competitive inhibitor at a lipid-water interface.

Animals↗
PubMed1992

Homo- and heteronuclear NMR studies of the human retinoic acid receptor beta DNA-binding domain: sequential assignments and identification of secondary structure elements.

An 80 amino acid polypeptide corresponding to the DNA-binding domain (DBD) of the human retinoic acid receptor beta (hRAR-beta) has been studied by 1H homonuclear and 15N-1H heteronuclear two- and three-dimensional (2D and 3D) NMR spectroscopy. The polypeptide has two putative zinc fingers homologous to those of the receptors for steroid and thyroid hormones and vitamin D3. The backbone 1H resonances as well as over 90% of the side-chain 1H resonances have been assigned by 1H homonuclear 2D techniques except for the three N-terminal residues. The assignments have been confirmed further by means of 15N-1H heteronuclear 3D techniques, which also yielded the assignments of the 15N resonances. Additionally, stereospecific assignments of methyl groups of five valine residues were made. Sequential and medium-range NOE connectivities indicate several elements of secondary structure including two alpha-helices consisting of residues E26-Q37 and Q61-E70, a short antiparallel beta-sheet consisting of residues P7-F9 and S23-C25, four turns consisting of residues P7-V10, I36-N39, D47-C50, and F69-G72, and several regions of extended peptide conformation. Similarly, two helices are found in the glucocorticoid receptor (GR) DBD in solution [Härd et al. (1990) Science 249, 157-160] and in crystal [Luisi et al. (1991) Nature 352, 497-505], and in the estrogen receptor (ER) DBD in solution [Schwabe et al. (1990) Nature 348, 458-461], although the exact positions and sizes of the helices differ somewhat. Furthermore, long-range NOEs suggest the existence of a hydrophobic core formed by the two helices.

Amino Acid Sequence↗
PubMed1992

Synthesis of isotope labeled oligonucleotides and their use in an NMR study of a protein-DNA complex.

The synthesis of an oligonucleotide labeled with 13C at the thymine methyl and 15N at the exocyclic amino groups of the cytosines is described. 13CH3I and 15NH4OH were used as sources of the labels. The labeled oligonucleotide was characterized by several NMR techniques. The duplex possesses a labeled functional group in the major groove at every base pair which makes it a very suitable probe for the study of sequence-specific protein-DNA interaction. The labeled thymine methyl group facilitates the detection of hydrophobic contacts with aliphatic side-chains of proteins. This is demonstrated in an NMR study of a complex between the glucocorticoid receptor DNA-binding domain and the labeled oligomer, which revealed a hydrophobic contact between a thymine methyl group and the methyl groups of a valine residue. There are indications for small differences between the solution structure the X-ray structure of the complex.

Animals↗
PubMed1992

Application of 2D and 3D NMR experiments to the conformational study of a diantennary oligosaccharide.

By the application of homonuclear 3D NOE-HOHAHA and heteronuclear 3D HMQC-NOE experiments in studies of complex oligosaccharides, NOEs can be investigated which are hidden in conventional 2D NOE spectra. In the 3D NOE-HOHAHA spectrum omega 3 cross sections were considered to be the most suitable for assignment of NOEs. Alternatively, these cross sections could be measured separately in selective 2D HOHAHA-NOE spectroscopy. The advantages and limitations of the 2D alternative are compared with those of the 3D NOE-HOHAHA approach. In 3D HMQC-NOE spectroscopy the larger chemical shift displacement of the carbon spectrum with respect to the proton spectrum can be used to unmask NOEs hidden in the bulk region. If the extra proton dimension is not needed, 2D HMQC-NOE is a good alternative. The suitability of 2D and 3D NOE-HOHAHA and HMQC-NOE experiments for the estimation of proton-proton distances is demonstrated by comparing the results of these experiments on a diantennary asparagine-linked oligosaccharide with those of a conventional 2D NOE experiment. NOEs identified in the 2D and 3D NOE-HOHAHA as well as HMQC-NOE experiments, so far not identified or not quantified in 2D NOE experiments, are discussed in relation to each glycosidic linkage. The flexibility of the Man alpha (1-3)Man linkage is demonstrated, confirming the existence of an ensemble of conformations for this linkage.

Carbohydrate Conformation↗
PubMed1992

The structure of the human retinoic acid receptor-beta DNA-binding domain determined by NMR.

The solution structure of the DNA-binding domain (DBD) of the human retinoic acid receptor-beta (hRAR-beta) has been determined by nuclear magnetic resonance (NMR) spectroscopy and distance geometry (DG). The assignments of 1H and 15N resonances were carried out by the use of 1H homonuclear and 15N-1H heteronuclear two- and three-dimensional NMR experiments. The structure of RAR DBD has been obtained on the basis of distance constrains derived from NMR experiments. The structure shows that two "zinc-finger" domains of the protein are followed by two perpendicular alpha-helices and a short beta-sheet near the N-terminus. Apolar residues in both helices form a hydrophobic core. Binding models of RAR DBD to its inverted and direct repeat response elements have been constructed based on this three-dimensional structure.

Carrier Proteins↗
PubMed1992

1H NMR studies on the oxidized ferredoxin from Clostridium pasteurianum.

The 8Fe-8S ferredoxin from Clostridium pasteurianum was investigated by 1D and 2D 1H NMR. Spectra of a well-structured, full native preparation of the oxidized protein in 1 M NaCl at pH 8.0 are presented. Assignments of non-isotropically shifted resonances in the diamagnetic region of the spectrum, namely those of the unique aromatic residues F30 and Y2, are presented for the first time.

Clostridium↗
PubMed1991

1H NMR studies of DNA recognition by the glucocorticoid receptor: complex of the DNA binding domain with a half-site response element.

The complex of the rat glucocorticoid receptor (GR) DNA binding domain (DBD) and half-site sequence of the consensus glucocorticoid response element (GRE) has been studied by two-dimensional 1H NMR spectroscopy. The DNA fragment is a 10 base-pair oligonucleotide, 5'd(GCTGTTCTGC)3'.5'd-(GCAGAACAGC)3', containing the stronger binding GRE half-site hexamer, with GC base pairs at each end. The 93-residue GR-DBD contains an 86-residue segment corresponding to residues 440-525 of the rat GR. Eleven NOE cross peaks between the protein and DNA have been identified, and changes in the chemical shift of the DNA protons upon complex formation have been analyzed. Using these protein-DNA contact points, it can be concluded that (i) the "recognition helix" formed by residues C460-E469 lies in the major groove of the DNA; (ii) the GR-DBD is oriented on the GRE half-site such that residues A477-D481, forming the so-called D-loop, are available for protein-protein interaction in the GR-DBD dimer on the intact consensus GRE; and (iii) the 5-methyl of the second thymine in the half-site and valine 462 interact, confirming indirect evidence [Truss et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7180-7184; Mader et al. (1989) Nature 338, 271-274] that both play an important role in GR-DBD DNA binding. These findings are consistent with the model proposed by Härd et al. [(1990) Science 249, 157-160] and the X-ray crystallographic complex structure determined by Luisi et al. [(1991) Nature 352, 497-505].

Amino Acid Sequence↗
PubMed1991

The solution structure of a monomeric insulin. A two-dimensional 1H-NMR study of des-(B26-B30)-insulin in combination with distance geometry and restrained molecular dynamics.

The solution conformation of des-(B26-B30)-insulin (DPI) has been investigated by 1H-NMR spectroscopy. A set of 250 approximate interproton distance restraints, derived from two-dimensional nuclear Overhauser enhancement spectra, were used as the basis of a structure determination using distance geometry (DG) and distance-bound driven dynamics (DDD). Sixteen DG structures were optimized using energy minimization (EM) and submitted to short 5-ps restrained molecular dynamics (RMD) simulations. A further refinement of the DDD structure with the lowest distance errors was done by energy minimization, a prolonged RMD simulation in vacuo and a time-averaged RMD simulation. An average structure was obtained from a trajectory generated during 20-ps RMD. The final structure was compared with the des-(B26-B30)-insulin crystal structure refined by molecular dynamics and the 2-Zn crystal structure of porcine insulin. This comparison shows that the overall structure of des-(B26-B30)-insulin is retained in solution with respect to the crystal structures with a high flexibility at the N-terminal part of the A chain and at the N-terminal and C-terminal parts of the B chain. In the RMD run a high mobility of Gly A1, Asn A21 and of the side chain of Phe B25 is noticed. One of the conformations adopted by des-(B26-B30)-insulin in solution is similar to that of molecule 1 (Chinese nomenclature) in the crystal structure of porcine insulin.

Amino Acid Sequence↗
PubMed1991

Identification of the metal coordinating residues in the DNA binding domain of the glucocorticoid receptor by 113Cd-1H heteronuclear NMR spectroscopy.

Two-dimensional 1H-113Cd HSQC and relay HSQC experiments were performed on the 113Cd substituted DNA binding domain of the rat glucocorticoid receptor. The results of these experiments combined with sequence-specific assignments allowed the identification of all coordinating cysteines. It was found that C495 and not C500 is the fourth coordinating cysteine in the second zinc-finger. A signal at approximately 2 ppm previously assigned to a epsilon-CH3 of a methionine residue coordinating to a third, weakly bound, cadmium ion, was identified as the C443 beta proton ligating to the metal ion in the first zinc-finger. No indications were found for the presence of a previously suggested third metal ion binding site.

Amino Acid Sequence↗
PubMed1991

Two-dimensional 1H-NMR studies of phospholipase-A2-inhibitor complexes bound to a micellar lipid-water interface.

One- and two-dimensional NMR studies were performed on the complexes of porcine pancreatic phospholipase A2 with substrate analogs bound to a micellar lipid-water interface of fully deuterated dodecylphosphocholine. The interactions between the inhibitor and the enzyme were localized by comparison of the two-dimensional NOE spectra recorded for the enzyme-inhibitor complex using both protonated and selectively deuterated inhibitors. These experiments led us to the following conclusions for the phospholipase-A2-micelle complex: (i) the 38-kDa phospholipase A2 complex gives NMR spectra with relatively narrow lines, which is indicative of high mobility of the enzyme; (ii) the residues Ala1, Trp3, Phe63 and Tyr69 located in the interface recognition site, as well as Phe22, Tyr75, Phe106 and Tyr111 are involved in the micelle-binding process; (iii) when present on the micelle, phospholipase A2 is stereospecific for the inhibitor binding; (iv) the inhibitor, (R)-dodecyl-2-aminohexanol-1-phosphoglycol, binds stoichiometrically to phospholipase A2 with high affinity (Kd less than or equal to 10 microM); (v) the inhibitor binds in the active site of the enzyme, which is evidenced by large chemical-shift differences for Phe5, Ile9, Phe22, His48, Tyr52 and Phe106; (vi) the acyl chain of the inhibitor makes hydrophobic contacts (less than 0.4 nm) near Phe5, Ile9, Phe22 and Phe106. Comparison of our results on the enzyme-inhibitor-micelle ternary complex with the crystal structure of the enzyme-inhibitor complex [Thunnissen, M. M. G. M., AB, E., Kalk, K. H., Drenth, J., Dijkstra, B. W., Kuipers, O. P., Dijkman, R., de Haas, G. H. & Verheij, H. M. (1990) Nature 347, 689-691] shows that the mode of inhibitor binding is similar.

Animals↗