Papers by Agustin Kintanar
Nature Structural Biology, 1994
Bookmarks Related papers MentionsView impact
Archives of Biochemistry and Biophysics, 1998
Bookmarks Related papers MentionsView impact
Plant Science, Mar 1, 1996
ABSTRACT The EMB-1 protein is a highly hydrophilic protein of unknown function that accumulates i... more ABSTRACT The EMB-1 protein is a highly hydrophilic protein of unknown function that accumulates in embryos of carrot before seed desiccation. To investigate the structure of this protein, it was expressed in E. coli, and purified to near homogeneity by Q-Sepharose anion exchange chromatography and ultrafiltration. The purified protein was characterized by 1H nuclear magnetic resonance (NMR) spectroscopy under both aqueous conditions and conditions simulating desiccation. The NMR results reveal poor chemical shift dispersion, rapid chemical exchange of the amide backbone protons, unexpectedly narrow linewidths and the general absence of nuclear Overhauser effects. Together, these results indicate the EMB-1 protein has no defined secondary or tertiary structure in solution and the polypeptide backbone of the EMB-1 protein is flexible on a sub-nanosecond time scale.
Bookmarks Related papers MentionsView impact
Biochemistry Usa, 1994
The structures of in vitro transcribed Escherichia coli tRNA(Val), which lacks base modifications... more The structures of in vitro transcribed Escherichia coli tRNA(Val), which lacks base modifications, and the native tRNA, which contains them, are very similar in the presence of excess Mg2+ (Kintanar, Yue, and Horowitz, unpublished results). To further probe the effects of base modifications on the structure of tRNA, the Mg2+ ion dependence of the downfield region of the 1H NMR spectrum of in vitro transcribed E. coli tRNA(Val) in aqueous phosphate buffer was investigated. The spectra indicate a remarkable conformational change in unmodified E. coli tRNA(Val) coincident with binding or release of Mg2+. Assignment of the imino proton resonances in the low Mg2+ form of the tRNA transcript allows a detailed description of the conformational change. There is near total disruption of the D stem and tertiary interactions in the absence of bound Mg2+. A new strong interaction between the U67-A6 base pair and the G50-U64 wobble pair is observed, indicating a substantial structural rearrangement at the junction of the acceptor and T stems. The binding constants of the strong Mg2+ binding sites in the D loop and near the D stem in unmodified tRNA(Val) are at least 2 orders of magnitude less than in tRNAVal containing base modifications. The metal ion binding site in the anticodon loop is somewhat stronger than metal ion binding sites in the D loop and stem in unmodified tRNA(Val), but it is still weaker than all strong Mg2+ binding sites in native tRNA(Val). Thus, one role of the base modifications found in tRNA is to stabilize or strengthen the Mg2+ binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)
Bookmarks Related papers MentionsView impact
Nucleic Acids Research, 1987
Bookmarks Related papers MentionsView impact
Journal of the American Chemical Society, 1986
Bookmarks Related papers MentionsView impact
The first deuterium NMR spectra of an individual membrane protein, bacteriohodopsin in the purple... more The first deuterium NMR spectra of an individual membrane protein, bacteriohodopsin in the purple membrane of Halobacterium halobium R1 has been obtained. Biosynthetic isotopic enrichment with (gamma-2H6) valine and high field Fourier transform operation permitted rapid data acquisition on intact membranes, including measurement of relaxation times. At some temperatures high quality spectra could be obtained in less than 1 s.
Bookmarks Related papers MentionsView impact
(8) Lovinger, AJ; Davis, DD Polym. Commun. 1985,26, 322. (9) Attwood, T. E.; Dawson, PC; Freeman,... more (8) Lovinger, AJ; Davis, DD Polym. Commun. 1985,26, 322. (9) Attwood, T. E.; Dawson, PC; Freeman, JL; Hoy, L. R.; Rose, JB; Staniland, P. A. Polymer 1981, 22, 1096. (10) Bishop, MT; Karasz, F. E.; Russo, F. E.; Langley, K. H. Macromolecules 1985, 18, 86. (11) Gaur, U.; ...
Bookmarks Related papers MentionsView impact
Biochemistry Usa, 1997
Bookmarks Related papers MentionsView impact
The Journal of biological chemistry, Jan 10, 1981
We have obtained the first deuterium NMR spectra of individual types of aromatic amino acids in a... more We have obtained the first deuterium NMR spectra of individual types of aromatic amino acids in a defined membrane protein, bacteriorhodopsin, in the photosynthetic purple membrane of Halobacterium halobium R1. Isotopic labeling and high field (8.5 Tesla) operation permitted relatively rapid data acquisition at a variety of temperatures. At the temperature of growth (37 degrees C), we find that all 7 tryptophan residues are rigid on the time scale of the NMR experiment (approximately 10(-5) s), except for likely librational motions of approximately 10 degrees amplitude. By contrast, nearly all (9 +/- 2) of the 11 tyrosines and (13 +/- 2) 13 phenylalanines undergo rapid (greater than 10(5) s-1) 2-fold rotational flips about C gamma-C zeta, causing formation of line shapes dominated by effectively axially asymmetric (asymmetry parameter eta = 0.66) deuteron electric field gradient tensors. On cooling the phenylalanine- and tyrosine-labeled samples to approximately -30 degrees C, all s...
Bookmarks Related papers MentionsView impact
The Journal of biological chemistry, Jan 10, 1981
We have obtained the first deuterium NMR spectra of an individual membrane protein, bacteriorhodo... more We have obtained the first deuterium NMR spectra of an individual membrane protein, bacteriorhodopsin in the purple membrane of Halobacterium halobium R1. Biosynthetic isotopic enrichment with [gamma-2H6]valine and high field Fourier transform operation permitted rapid data acquisition on intact membranes, including measurement of relaxation times. At some temperatures high quality spectra could be obtained in less than 1 s. [U-14C]Valine tracer studies indicate that less than or equal to 2% of valine added to the growth medium is broken down and incorporated into other membrane constituents. The NMR results indicate that the valine side chain is a rather rigid structure. Motion about C alpha-C beta is slow (less than 10(5) s-1) at growth temperature, While motion about C beta-C gamma is as expected fast (much greater than 10(5) s-1) at all accessible temperatures. The activation energy for methyl group rotation from spin-lattice relaxation data between -75 and 53 degrees C is appro...
Bookmarks Related papers MentionsView impact
Proceedings of the National Academy of Sciences, 1991
Bookmarks Related papers MentionsView impact
Plant Science, 1996
ABSTRACT The EMB-1 protein is a highly hydrophilic protein of unknown function that accumulates i... more ABSTRACT The EMB-1 protein is a highly hydrophilic protein of unknown function that accumulates in embryos of carrot before seed desiccation. To investigate the structure of this protein, it was expressed in E. coli, and purified to near homogeneity by Q-Sepharose anion exchange chromatography and ultrafiltration. The purified protein was characterized by 1H nuclear magnetic resonance (NMR) spectroscopy under both aqueous conditions and conditions simulating desiccation. The NMR results reveal poor chemical shift dispersion, rapid chemical exchange of the amide backbone protons, unexpectedly narrow linewidths and the general absence of nuclear Overhauser effects. Together, these results indicate the EMB-1 protein has no defined secondary or tertiary structure in solution and the polypeptide backbone of the EMB-1 protein is flexible on a sub-nanosecond time scale.
Bookmarks Related papers MentionsView impact
Nucleic Acids Research, 1987
The resonances of all the non-exchangeable protons (except 5'H and 5"H) of d(CGA... more The resonances of all the non-exchangeable protons (except 5'H and 5"H) of d(CGAAAAATCGG) + d(CCGATTTTTCG), a putatively bent DNA duplex, have been assigned using 1H two-dimensional nuclear magnetic resonance methods. The nuclear Overhauser effect data indicate an overall B-form structure for this double-helical DNA undecamer. However, several features of the NMR data such as some unusually weak C8/C6 proton to C1' proton NOE cross-peaks, the presence of relatively intense C2H to C1'H NOE cross-peaks, and unusual chemical shifts of some 2", 2', and 1' protons suggest a substantial perturbation of the helix structure at the junctions and along the length of the tract of A residues. These structural deviations are considered in terms of models of DNA bending.
Bookmarks Related papers MentionsView impact
Macromolecules, 1986
(8) Lovinger, AJ; Davis, DD Polym. Commun. 1985,26, 322. (9) Attwood, T. E.; Dawson, PC; Freeman,... more (8) Lovinger, AJ; Davis, DD Polym. Commun. 1985,26, 322. (9) Attwood, T. E.; Dawson, PC; Freeman, JL; Hoy, L. R.; Rose, JB; Staniland, P. A. Polymer 1981, 22, 1096. (10) Bishop, MT; Karasz, F. E.; Russo, F. E.; Langley, K. H. Macromolecules 1985, 18, 86. (11) Gaur, U.; ...
Bookmarks Related papers MentionsView impact
Journal of the American Chemical Society, 1988
Bookmarks Related papers MentionsView impact
Journal of the American Chemical Society, 1986
Bookmarks Related papers MentionsView impact
Journal of the American Chemical Society, 1990
Abstract: Solid-state deuterium NMR spectroscopy has been used to characterize the dynamics of th... more Abstract: Solid-state deuterium NMR spectroscopy has been used to characterize the dynamics of the furanose rings of A5 and A6 (and by symmetry AI7 and AIS) in the self-complementary DNA dodecamer duplex [d (CGCGAATTCGCG)],, which contains the ...
Bookmarks Related papers MentionsView impact
Journal of Molecular Biology, 1992
To complete assignment of the 19F nuclear magnetic resonance (NMR) spectrum of 5-fluorouracil-sub... more To complete assignment of the 19F nuclear magnetic resonance (NMR) spectrum of 5-fluorouracil-substituted Escherichia coli tRNA(Val), resonances from 5-fluorouracil residues involved in tertiary interactions have been identified. Because these assignments could not be made directly by the base-replacement method used to assign 5-fluorouracil residues in loop and stem regions of the tRNA, alternative assignment strategies were employed. FU54 and FU55 were identified by 19F homonuclear Overhauser experiments and were then assigned by comparison of their 19F NMR spectra with those of 5-fluorouracil-labeled yeast tRNA(Phe) mutants having FU54 replaced by adenine and FU55 replaced by cytosine. FU8 and FU12, were assigned from the 19F NMR spectrum of the tRNA(Val) mutant in which the base triple G9-C23-G12 substituted for the wild-type A9-A23-FU12. Although replacement of the conserved U8 (FU8) with A or C disrupts the tertiary structure of tRNA(Val), it has only a small effect on the catalytic turnover number of valyl-tRNA synthetase, while reducing the affinity of the tRNA for enzyme. Analysis of the 19F chemical shift assignments of all 14 resonances in the spectrum of 5-fluorouracil-substituted tRNAVal indicated a strong correlation to tRNA secondary and tertiary structure. 5-Fluorouracil residues in loop regions gave rise to peaks in the central region of the spectrum, 4.4 to 4.9 parts per million (p.p.m.) downfield from free 5-fluorouracil. However, the signal from FU59, in the T-loop of tRNA(Val), was shifted more than 1 p.p.m. downfield, to 5.9 p.p.m., presumably because of the involvement of this fluorouracil in the tertiary interactions between the T and D-loops. The 19F chemical shift moved upfield, to the 2.0 to 2.8 p.p.m. range, when fluorouracil was base-paired with adenine in helical stems. This upfield shift was less pronounced for the fluorine of the FU7.A66 base-pair, located at the base of the acceptor stem, an indication that FU7 is only partially stacked on the adjacent G49 in the continuous acceptor stem/T-stem helix. An unanticipated finding was that the 19F resonances of 5-fluorouracil residues wobble base-paired with guanine were shifted 4 to 5 p.p.m. downfield of those from fluorouracil residues paired with A. In the 19F NMR spectra of all fluorinated tRNAs studied, the farthest downfield peak corresponded to FU55, which replaced the conserved pseudouridine normally found at this position.
Bookmarks Related papers MentionsView impact
Biochimie, 1994
Transfer RNA transcribed in vitro lacks the base modifications found in native tRNA. To understan... more Transfer RNA transcribed in vitro lacks the base modifications found in native tRNA. To understand the effect of base modifications on the structure of tRNA, the downfield region of the 1H NMR spectrum of in vitro transcribed E coli tRNAVal in aqueous phosphate buffer in the presence of excess Mg2+ was investigated. The resonances of all imino protons involved in hydrogen bonds in the helical stem regions and in tertiary interactions were assigned using two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) and one-dimensional difference nuclear Overhauser effect (NOE) methods. In addition, some aromatic C2 and C8 proton resonances as well as one amino proton resonance were assigned. The chemical shifts of the assigned resonances of unmodified E coli tRNAVal were compared with those of the native tRNA molecule under similar solution conditions. The similarity of the NMR data for unmodified and modified tRNA indicates that the in vitro transcribed tRNA has nearly the same solution structure as the native molecule in the presence of excess Mg2+. The only significant differences were the chemical shifts of resonances corresponding to protons in (or interacting with) bases, indicating the possibility of local structural perturbations. The thermal stability of E coli modified and unmodified tRNAVal in the presence of Mg2+ was also investigated by analyzing the temperature dependence of the imino proton spectra. Several tertiary interactions involving modified nucleosides in native E coli tRNAVal are less stable in the absence of base modifications.
Bookmarks Related papers MentionsView impact
Uploads
Papers by Agustin Kintanar