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R G Shulman

Publications and source records attributed to R G Shulman.

At least 217 records · Page 12Linked to original sources
PubMed1976

A study of secondary and tertiary solution structure of yeast tRNA(Asp) by nuclear magnetic resonance. Assignment of G.U ring NH and hydrogen-bonded base pair proton resonances.

The 270-MHz spectra of yeast tRNA(Asp) in H2O solutions containing Mg2+ show clearly resolved resonances in the region from -15 to -9.5 ppm. Resonances between -15 and -11.5 ppm from the hydrogen-bonded protons of the acceptor stem and anticodon arm decrease in intensity with increasing temperature and disappear by 75 degrees C. Simultansously, four well-resolved resonances between -11.2 and -10.3 ppm also decrease in intensity and disappear. Because of this behavior and their positions these resonances have been assigned to the four ring NH protons of G.U base pairs 5 and 30 in the acceptor stem and anticodon arm which are thereby shown not to be hydrogen bonded by normal Watson-Crick hydrogen bonds. The five G.C base pair resonances of the T psi C arm remain visible above 70 degrees C after all other resonances have disappeared. The high-temperature tRNA spectrum agrees well with that of the isolated T psi C hairpin and CCA half-molecule fragments, each of which contains the same five hydrogen-bonded proton resonances. The root-mean-square error between the observed and calculated resonance positions for the hydrogen-bonded base pair protons of these three arms is 0.19 ppm. The dihydrouridine stem is expected to have two A.U Watson-Crick base pairs and no B.C base pairs. However, it does not contribute any hydrogen-bonded resonances to the nuclear magnetic resonance (NMR) spectrum below -11.5 ppm. This suggests that even at 35 degrees C this helix is not hydrogen bonded in a normal manner. In the region below -11.4 ppm there are three additional proton resonances melting earlier than the rest which cannot be assigned to a particular helix of the cloverleaf. We suggest that these resonances arise from hydrogen-bonded protons involved in stabilizing tertiary structure.

Binding Sites↗
PubMed1976

Structure-function relations in hemoglobin as determined by x-ray absorption spectroscopy.

Conclusions concerning the structure around the iron atom in oxy- and carbonmonoxyhemoglobin have been obtained by fluorescent x-ray absorption studies. The bis-imidazole heme complex was used as a model system of known structure. The ligated forms of hemoglobin, and cytochrome c at high pH, gave spectra which were very similar to the bis-imidazole complex, where the average Fe-N bond distance is known to be 1.98 A. By comparison it was possible to determine that the average Fe-N bond distances were 1.99 A in oxyhemoglobin, 1.98 A in carbonmonoxyhemoglobin, and 1.98 A in cytochrome c at pH less than 10.5, with an experimental accuracy of +/-0.02 A. An experimental comparison between oxy- and deoxyhemoglobin A showed much larger spectral changes than amongst the ligated forms. A comparison was made between the low oxygen affinity form of deoxy HbA and the high affinity form of doexy Hb Kempsey (alpha2beta992 Asp leads to Asn). All the spectral features coincided, allowing us to conclude that the average iron-ligand bond differences must be less than or equal to 0.02 A. Since the strain energy is proportional to the square of this displacement, we show that the strain energy at the iron is less than or equal to 4 X 10(-3) eV. This is negligible compared to the difference of binding energy of the high and low affinity forms, which is 0.15 eV, showing that the energies responsible for the increase of oxygen affinity are not localized at the heme.

Carboxyhemoglobin↗
PubMed1976

Physical studies of denatured tRNA2Glu from Escherichia coli.

We have examined the 270 MHz nuclear magnetic resonance spectrum and relaxation kinetic behavior of tRNA2Glu (E. coli) in the absence of Mg++, a condition which produces an inactive form of this tRNA. The results show that the denatured form has about five fewer proton resonances in the region from -12 to -15 ppm. Relaxation kinetic measurements reveal that the denatured conformer contains three separately melting helices. The results support a model in which the tertiary structure and dihydrouridine helix characteristic of the native form are unfolded in the denatured state, and are replaced by an altered tertiary structure. The acceptor stem, anticodon, and TpsiC helices are intact in this model for the denatured conformation. The optical changes that accompany melting of the denatured tertiary structure are faster than 10 musec.

Cations, Divalent↗
PubMed1975

Correlation between quaternary structure and ligand dissociation kinetics for fully liganded hemoglobin.

The quaternary structures of fully liganded adult hemoglobin and hemoglobin Kansas (alpha2beta2 102 Asn-thr) bound by carbon monoxide or nitric oxide were spectroscopically characterized using high-resolution nuclear magnetic resonance (NMR) and ultraviolet circular dichroism (CD). The spectral markers used for the quarternary transition were the line in the NMR spectrum in H2O-14 ppm downfield from 2,2-dimethyl-2-silapentane-5-sulfonate and the negative peak at 285 nm in the ultraviolet CD spectrum. In the nitrosyl derivatives, these two structural markers were compared with the electron paramagnetic resonance (EPR) spectrum at room temperature for the purpose of correlating structural changes in the protein with changes at the heme...

Binding Sites↗
PubMed1975

Determination of the iron-sulfur distances in rubredoxin by x-ray absorption spectroscopy.

The high intensity x-ray flux from the synchrotron radiation at the Stanford Synchroton Radiation Project has been used to study the extended x-ray absorption fine structure (EXAFS) of the iron-sulfur protein Peptococcus aerogenes rubredoxin. Absorption measurements were made from 7080 eV, which is below the K-edge of iron, to about 650 eV above the edge and structure was obtained over the entire region. By means of a model iron-sulfur compound for evaluating the phase shifts, the variation of the absorption above the edge of lyophilized, oxidized rubredoxin was converted to iron-sulfur distances. The data were fitted with a least squares program to a model in which three distances R3 were kept equal and the fourth R1 was allowed to differ. The mean square error was constant over a region of this parameter space, becoming twice as large at R3 = 2.217, R1 = 2.389 and R3 = 2.268, R1 = 2.108 A. These values, which are the extreme differences allowed by the present data, are definitely closer to being equal than those found by the determination of the x-ray diffraction crystal structure of the similar protein from Clostridium pasteurianum. However, the average distance from our experiment is in excellent agreement with the average distance from the crystal structure determination. Preliminary EXAFS measurements were also made on the oxidized rubredoxin in solution at pH 7.0. The spectra were unchanged, indicating that the average iron-sulfur distance change is less than 0.02 A. Upon reduction the average iron-sulfur bond length increased by about 0.05 A. Since the EXAFS measurements can give accurate determinations of distances in proteins both in crystals and solution, the technique should be widely applicable.

Binding Sites↗
PubMed1975

High resolution 31P nuclear magnetic resonance studies of intact yeast cells.

High resolution 31P nuclear magnetic resonance (NMR) spectra at 145.7 MHZ are presented for intact yeast cells. Several peaks are resolved and assigned. They include the middle phosphate peaks from long chain or cyclic polyphosphates. Our results are consistent with the suggestion that these polyphosphates act as a phosphate store in the cell. We have also been able to measure cytoplasmic pH using the orthophosphate peak inside the cell, as compared with outside the cell. The results show that yeast cells maintain their cytoplasmic pH around 6.3. This value is considerably higher than the acidic extracellular pH at which they normally live. These preliminary results indicate that 31P NMR at 145.7 MHZ can be a rapid, informative, and non-invasive method for probing biochemical events within living cells.

Hydrogen-Ion Concentration↗
PubMed1975

Tertiary hydrogen bonds in the solution structure of transfer RNA.

The high resolution nuclear magnetic resonance (NMR) spectra of hydrogen-bonded protons in four tRNAs have been studied at 270 MHz. The relative intensity of the resonances between -11 ppm and -15 ppm of Escherichia coli tRNA1-Va1 indicate that there are 26 plus or minus 3 protons, while only 20 are expected from secondary structure Watson-Crick hydrogen bonds inthe cloverleaf structure. Several possible candidates for these extra resonances are suggested by tertiary interactions observed in recent crystallographic studies. Of the four tRNAs studied, three, e.g., E. coli tRNA1Va1, E. coli tRNA-Arg and E. coli tRNA-Phe have one "GU pair" in their cloverleaf structure, while the fourth, yeast tRNA-Asp,has three "GU pairs" and one "G pair". Correlating these with the NMR spectra in the -10 ppm to -11 ppm region allows us to conclude that the "GU pairs" are not hydrogen-bonded by tautomerization to the lactim form. At the very low field region, near -14.9 ppm, the three E. coli tRNAs show a single resonance which is attributed to the 4-thiouracil 8 to adenine 14 hydrogen bond of the tertiary structure, by analogy with the recent crystal structure of yeast tRNA-Phe. This assignment is confirmed by the disappearance of this resonance after treatment with cyanogen bromide.

Arginine↗
PubMed1975

31P magnetic resonance of tRNA.

We have observed well-resolved 31P resonances at 65 kG (109 MHz) in solutions of Escherichia coli tRNAGlu and yeast tRNAPhe. One resolved resonance, identified as the terminal phosphate, titrates with a pK = 6.35. Upon melting the yeast tRNAPhe in 0.1 M NaCl, without Mg++, the resolved peaks broaden and disappear in the vicinity of 35 degrees while the central cluster narrows drastically, shifts slightly, and loses its structure. Addition of Mg++ shifts one resolved peak and changes the shape of the cluster. The 31P lines are broader when observed at 65 kG than at 24 kG. The broadening is shown to come from chemical shift anisotropy which is estimated to be about 140 ppm.

Escherichia coli↗
PubMed1974

13C high-resolution nuclear magnetic resonance studies of enzyme-substrate reactions at equilibrium. Substrate studies of chymotrypsin-N-acetyltyrosine semicarbazide complexes.

N-Acetyl-L-tyrosine semicarbazide is hydrolyzed by chymotrypsin (EC 3.4.21.1) to N-acetyl-L-tyrosine and semicarbazide. If a high concentration of semicarbazide is present, the equilibrium for the reaction can be shifted from hydrolysis to synthesis. Using N-acetyl-L-[(13)C]tyrosine enriched at the carboxyl carbon and high concentrations of semicarbazide hydrochloride, we have studied the enzyme-substrate complex of N-acetyl-L-[(13)C]tyrosine semicarbazide and chymotrypsin A(delta) by (13)C nuclear magnetic resonance. We observe no shift within the experimental accuracy of +/-0.05 ppm as the fraction of substrate bound is changed from 0.17 to 0.70. Since E + S right arrow over left arrow ES is in fast exchange on the nuclear magnetic resonance time scale, it is possible to show that when the substrate is bound to the enzyme in the Michaelis complex, the (13)C resonance is shifted less than 0.1 ppm, indicating that negligible substrate strain occurs in this complex at the site of enzymatic attack. These experiments demonstrate the application of nuclear magnetic resonance to the study of particular states along the reaction pathway for enzyme-substrate reactions at equilibrium.

Carbon Isotopes↗
PubMed1974

Assignment of the hydrogen bonded proton resonances in (Escherichia coli) tRNAGlu by sequential melting.

The 300 MHz nuclear magnetic resonance peaks from the hydrogen bonded protons in (Escherichia coli) tRNA(Glu) in Mg(++) solutions have been measured at temperatures between 25 degrees and 74 degrees . Between 25 degrees and 49 degrees four resonances broaden, three of which are assigned to the hU arm and one to a G.C pair of the tertiary structure. By 64 degrees these four resonances have disappeared and the nuclear magnetic resonance spectrum is fitted very well by a computer simulation based upon resonances from the acceptor, T-Psi-C, and anticodon arms. At 66 degrees the resonances from the T-Psi-C arm are lost and at 74 degrees only those from the anticodon are left. All 20 resonances expected from the cloverleaf model have been assigned by comparing the calculated positions of resonances in a particular arm with the stepwise loss of intensity with temperature. The root mean square error between calculated and observed positions is 0.17 ppm. Resonances at the end of helical regions which are sensitive to stacking beyond the helix allow us to conclude that the acceptor arm is stacked upon the T-Psi-C in a regular helix and that G [unk]73 is stacked upon base pair [unk]1-72 but the hU and anticodon arms are not stacked in a regular helix upon the intervening base A [unk]27.

Base Sequence↗
PubMed1974

Spectral-kinetic heterogeneity in reactions of nitrosyl hemoglobin.

When NO replaces CO in hemoglobin A in the presence of inositol hexaphosphate, the time course is heterogeneous in contrast to stripped hemoglobin A, where it is homogeneous. If nitrosyl hemoglobin is mixed with inositol hexaphosphate in the stopped-flow apparatus, an extra spectral change is observed which is the cause of the spectral-kinetic heterogeneity in the CO replacement reaction. At wavelengths isosbestic for this extra spectral change, the time courses show an accelerating rate of CO dissociation. On the other hand, the same reaction for NES-des-Arg hemoglobin (hemoglobin reacted with N-ethylmaleimide and carboxypeptidase B) in the presence of inositol hexaphosphate is homogeneous and slow, and shows isosbesty. High-resolution nuclear magnetic resonance spectra indicate that adult nitrosyl hemoglobin in the presence of inositol hexaphosphate is in the low ligand affinity state, thus offering a structural basis for the acceleration observed in the rate of CO dissociation. Hemoglobin Kansas, in the presence of inositol hexaphosphate, which starts and finishes the reaction in the low affinity state, shows a rate of CO dissociation about nine times faster than stripped hemoglobin A. We conclude from these results that (i) the CO to NO replacement reaction can include a functionally important change in the overall conformation of fully liganded hemoglobin, depending on solution conditions and protein type; (ii) the extra spectral change observed for nitrosyl hemoglobin is not the functionally dominating conformational change, but is a secondary effect within the low ligand affinity protein structure; and (iii) the functional properties of heme ligands are largely controlled by two conformational states of the protein, as seen by nuclear magnetic resonance spectroscopy.

Carboxyhemoglobin↗