Biomedical subjects

R Kaptein

Publications and source records attributed to R Kaptein.

At least 109 records · Page 6Linked to original sources

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

Conformational analysis of a dinucleotide photodimer with the aid of the genetic algorithm.

The solution structure of the photodimer cis,syn-dUp[]dT is derived with the aid of the genetic algorithm. The conformational space available for the molecule is sampled efficiently using the computer program DENISE and tested against a set of constraints available from nmr experiments. The dominant conformation in solution found with this approach can be described by the following combinations of sugar-phosphate backbone torsion angles: epsilon(t), zeta(t), alpha(+), beta(-ac), and gamma(t). The conformation of the sugars and glycosidic torsion angles are S type and syn, respectively. The cyclobutane ring and pyrimidines are puckered. In addition, other conformations that exist in equilibrium with the first are found. It is concluded that the cyclobutane-pyrimidine system is rigid, whereas the sugar-phosphate backbone is flexible. The solution structures are compared with the crystal structure of the strongly related cyano-ethyl ester of cis,syn-dTp[]dT.

Algorithms↗

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

Flavin-sensitized photochemically induced dynamic nuclear polarization detection of pyrimidine dimer radicals.

A photochemically induced dynamic nuclear polarization (photo-CIDNP) study of carboxymethyllumiflavin-sensitized splitting of pyrimidine dimers has been carried out. In aqueous solution at high pH, an emission signal (delta 3.9 ppm) was observed from the dimer C(6)- and C(6')-protons of an N(1), N(1')-trimethylene-bridged thymine dimer (1). The dimer photo-CIDNP signal was seen only above pD 11.6 and was most intense at pD 12.9. Also observed were weak enhanced absorption signals from the product of splitting, trimethylenebis(thymine) (delta 1.7 and 7.2 ppm). In contrast, cis, syn-thymine dimer (3) gave no photo-CIDNP signals from the dimer. An enhanced absorption at 1.8 ppm, however, due to the product of splitting (thymine) was observed. It was found that dimer 1 and, to a lesser extent, dimer 3 quenched flavin fluorescence. An N(3),N(3')-dimethylated derivative of 1, however, failed to quench flavin fluorescence. Comparison of the pD profile of the dimer photo-CIDNP signal to the pKa values for thymidine dimer suggested that principally the dideprotonated dimer undergoes electron abstraction by the excited flavin.

Flavins↗

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↗

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↗

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

Interaction of apocytochrome c and derived polypeptide fragments with sodium dodecyl sulfate micelles monitored by photochemically induced dynamic nuclear polarization 1H NMR and fluorescence spectroscopy.

The topology of apocytochrome c, the heme-free precursor of the mitochondrial protein cytochrome c, was investigated in a lipid-associated form. For this purpose photochemically induced dynamic nuclear polarization 1H nuclear magnetic resonance (CIDNP 1H NMR) spectroscopy and quenching of tryptophan and tyrosine fluorescence by acrylamide were applied to an apocytochrome c-sodium dodecyl sulfate (SDS) micellar system. A pH titration of the chemical shifts of the histidine C2 proton resonances of apocytochrome c, using conventional 1H NMR, yielded pK(a)'s of 5.9 +/- 0.1 and 6.2 +/- 0.1, which were assigned to histidine-18 and -33 and histidine-26, respectively. In the presence of SDS micelles an average pK(a) of 8.1 +/- 0.1 was obtained for all histidine C2 protons. Photo-CIDNP enhancements of the histidine, tryptophan, and tyrosine residues, contained in the intact apocytochrome c and in chemically and enzymatically prepared fragments of the precursor, were reduced in the presence of SDS micelles. Similarly, the quenching of the tryptophan fluorescence of the polypeptides by acrylamide was diminished in the presence of SDS. These results indicate the aromatic residues studied are localized in the interface of the SDS micelle.

Animals↗

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

NMR studies of bipyrimidine cyclobutane photodimers.

Cyclobutane-type photodimers of dinucleoside monophosphates dCpdT, dTpdC and dTpdT were prepared by ultraviolet irradiation in the presence of acetophenone as photosensitizer. The cytosine-containing derivatives were found to deaminate forming uracil products. Using one- and two-dimensional NMR, the photoproducts were characterized as cis-syn and trans-syn cyclobutane photodimers. On the basis of NOE data the structures of the cis-syn and trans-syn products of dUpdT were determined using distance-geometry and restrained-energy-minimization methods. The cis-syn structures showed (high-ANTI/SYN)/high-ANTI glycosidic linkages while the trans-syn structures were in the SYN-ANTI region. The backbone conformations of both structures were in fair agreement with the coupling-constant-data. The trans-syn structures were found to be very rigid and similar in all three products. For the three cis-syn structures more conformational freedom and more variation among the three structures was observed.

Magnetic Resonance Spectroscopy↗