Dinshaw J. Patel

Nuclear magnetic resonance determination of ligand-induced conformational changes in myoglobin.

Abstract We report the results of a high-resolution proton nuclear magnetic resonance at 220 MHz of deoxymyoglobin and three diamagnetic complexes of myoglobin with oxygen, carbon monoxide and ethylisocyanide. We emphasize the resonances which are shifted from their normal positions by ring currents from aromatic rings, particularly the porphyrin ring, because these ring current shifts are very sensitive to changes of the relative co-ordinates of the observed protons and the ring. A specific resonance at −6.1 p.p.m., with intensity of about three protons, has been attributed to the protons of an aromatic residue, shifted upfield by the ring currents of the heme group. A possible origin of this resonance is Phe CD1. The observed change of this resonance of 0.2 p.p.m. upon oxygenation corresponds to a movement of a few tenths of an angstrom unit. Slightly larger shifts upon oxygenation of resonances attributed to methyl groups of aliphatic residues are interpreted in terms of movements of about 0.5 A. A comparison of the complete spectra of Mb ‡ and MbO2 shows that there are many small differences of 0.1 to 0.2 p.p.m. which indicate widespread small structural changes upon oxygenation. Differences in the ring-current-shifted resonances amongst various ligated forms including cyanoferrimyoglobin, indicate the existence of structural differences in the protein when the ligand is changed.

High resolution nuclear magnetic resonance study of base pairing in four purified transfer RNA molecules

Abstract The high resolution nuclear magnetic resonance spectra of hydrogen bonded protons in four purified tRNA molecules are reported. From the temperature and concentration dependence it is shown that these resonances arise from intramolecular hydrogen bonds.

Experimental and calculated conformational characteristics of the bicyclic heptapeptide phalloidin

Abstract Several conformations generated from approximate potential energy calculations are presented for the bicyclic heptapeptide phalloidin which are consistent with the conformation-dependent information obtained from proton nuclear magnetic resonance measurements performed on phalloidin in dimethylsulfoxide solution. In each conformation, the cysteine amide proton is intramolecularly hydrogen bonded, the tryptophan amide is internally buried and the methyl group of the alanine residue preceding tryptophan is shielded by the tryptophan ring. Thus, phalloidin appears to be a relatively rigid molecule in solution.

Bleomycin-A2 complexes with poly(dA-dT): A proton nuclear magnetic resonance study of the nonexchangeable hydrogens

Abstract Binding of bleomycin-A2 (Bleo-A2) to poly(dA-dT) in 100 mM sodium phosphate (pH 6.8) in D2O has been investigated by 1H NMR spectroscopy at 270 and 360 MHz. Significant spectral perturbations were observed only when the nucleic acid was in the duplex state. Of the Bleo-A2 resonances, the two bithiazole peaks exhibited the largest spectral shiffs and line broadening effects. The high field shift of these resonances was very small near room temperature and reached a maximum of about 0.27 ppm just below the thermal denaturation temperature of the nucleic acid (Tm = 60 ± 1°C). The temperature dependence of spectral perturbations may be accounted for by the formation of at least two types of Bleo-A2 complexes with the polynucleotide. Other perturbed resonances of bleomycin are the S-C H 3 and S-C H 2 of the terminal amine, the C H 2-N resonance of the bithiazole residue, and the C H (CH3)CO of the methylvaleric acid residue. The significantly perturbed resonances of the nucleic acid originate from the A(H-2), A(H-8), T(H-6) and one of the H-2′ hydrogens. Binding of the C-terminal tripeptide fragment of Bleo-A2 to poly(dA-dT) is accompanied by selective broadening of the bithiazole group. These experiments have identified potential loci of interaction on the Bleo-A2 and poly(dA-dT) molecules.

NMR studies of the structure and stability of the 1 : 2 actinomycin D · d-pG-C complex in aqueous solution

We describe NMR studies at superconducting fields which characterize aspects of the structure and stability of the 1 : 2 actinomycin-d-pG-C complex in solution as monitored at the Watson-Crick base pairs and backbone phosphate groups. Two guanine N1H resonances (12.17 and 11.66 ppm) are observed in the 360 MHz proton NMR spectra of the complex in water at -4 degrees C. These slowly exchangeable resonances, which demonstrate the presence of two Watson-Crick G + C base pairs in the complex, broaden in a sequential manner with increasing temperature. The terminal and internucleotide phosphates of both d-pG-C molecules are observable in the 145.7 MHz 31P spectra of the 1 : 2 actinomycin-d-pG-C complex at 0 degrees C. The internucleotide phosphate resonance at 1.905 ppm broadens prior to that at 2.385 ppm with increasing temperature, consistent with a sequential breakage of the G + C base pairs in the complex. The lifetime of the complex (4.5 +/- 0.5 X 10(-4) s, 33 degrees C) was deduced from the variation of the d-pG-D internucleotide 31P resonance line width on gradual addition of the antibiotic in solution.

Structure dynamics and energetics of the d(CGCGAATTCGCG) dodecamer duplex and its selectively modified analogues in solution

A great deal of our knowledge concerning the molecular forces that stabilise RNA structures has been obtained from systematic investigations of specially designed and synthesised ribo-oligonucleotides possessing the various structural features found in naturally-occurring RNA molecules. (Uhlenbeck et al., 1971; Craig et al., 1971; Martin et al., 1971; Porschke et al., 1973; Uhlenbeck et al., 1973; Gralla and Crothers 1973a; Borer et al., 1974; Breslauer et al., 1975). Fewer corresponding investigations have been conducted on DNA structures, primarily because of the lack of available deoxyoligomers. (Burd et al., 1975; Seising and Wells, 1979). Most early workers studied complementary homopolymers and self-complementary, alternating copolymers. (Wells and Wartell, 1974; Arnott et al., 1975; Wells et al., 1977; Arnott, 1977; Leslie et al., 1980). Although these studies were important, the lack of sequence heterogeneity caused the information content of these molecules to be somewhat limited. Furthermore, in the absence of controlled sequence modification, it was difficult to correlate a given experimental observation with a specific molecular event. Thus, the effects of mismatches, bulges and internal loops on the stability of DNA structures are just beginning to be understood (Dodgson and Wells, 1977; Early et al., 1977; Selsing et al., 1978; Wallace et al., 1979; Cornells et al., 1979; Haasnoot et al., 1980).

Sequence Dependence of DNA Conformation, Dynamics and Interactions in Solution

We have applied both distance-dependent nuclear Overhauser effect measurements to elucidate structural information and saturation recovery methods to evaluate hydrogen exchange kinetics at the individual base pair level for nucleic acids in aqueous solution. These experiments demonstrate a sequence dependence of the local conformation and dynamics of DNA in solution. We show that intermolecular nuclear Overhauser effect measurements can map out the complexation sites of antibiotics on the DNA helix and hydrogen exchange measurements can estimate the extent of kinetic stabilization at and adjacent to the ligand binding site.