R.G. Shulman

The molecular mechanism of thermal unfolding of Escherichia coli formylmethionine transfer RNA

Abstract The molecular mechanism of thermal unfolding of Escherichia coli tRNA fMet (in 0.17 m -NaCl without Mg 2+ ) has been elucidated by a combination of relaxation kinetics and proton nuclear magnetic resonance spectroscopy. We measured the n.m.r. ‡ spectrum of the hydrogen-bonded ring NH protons at different temperatures and found that the resonances assigned to each arm of the cloverleaf broaden and disappear together, yielding four distinct n.m.r. “melting” transitions. Temperature-jump measurements in the same solvent showed five co-operative melting transitions, varying in relaxation time from a few microseconds to ten milliseconds. The relaxation and n.m.r. measurements were correlated by the following model. When the lifetime of a hydrogen-bonded proton in a helix is five milliseconds, its n.m.r. line will be broadened to approximately twice its intrinsic low-temperature width and appear to “melt”. The helix dissociation time constants of the relaxation effects were extrapolated by the Arrhenius equation to lower temperatures where their values were five milliseconds. The correlation of extrapolated dissociation time constants with n.m.r. melting of specific helices allowed assignments of the structural basis for each relaxation effect. The results show that the principal path for the reversible thermal unfolding of tRNA 1 fMet under these solution conditions is first, transient opening of the dihydrouridine helix, followed by simultaneous melting of the dihydrouridine helix and a “tertiary” interaction, which does not correspond to a cloverleaf helix. The tertiary interaction is much less stable in tRNA 3 fMet , with T m lowered by 16 °C from tRNA 1 fMet . The sequence of melting steps at higher temperatures is the same in the two isoacceptors: first the TΨC helix melts, followed by the anticodon helix and finally the acceptor stem helix. Thermodynamic and kinetic parameters are reported for these steps. The method of sequential melting, combining n.m.r. and relaxation kinetic techniques, is a powerful procedure for elucidating RNA secondary structure. In addition, this method allows assignment of many hydrogen-bonded ring NH proton resonances that are unresolved in the low-temperature spectrum.

Electronic structure of cyanide complexes of hemes and heme proteins

Abstract A simple quantum mechanical model is introduced to interpret the shifts measured by nuclear magnetic resonance in a variety of low spin ( S = 1 2 ) ferric cyanide heme and heme protein complexes. An essential element of the model is its consideration of the pair of highest occupied π-orbitals, which are degenerate in D 4h symmetry. The observed shifts are related to the orientation of perturbations that lift the orbital degeneracy and to a coupling of the resulting pair of non-degenerate states. It is demonstrated that the variation of the shifts in different compounds can serve as a sensitive probe for the interaction of the heme group with its environment; of particular interest is the difference between the heme in solution and bonded to a globin molecule.

Allosteric interpretation of haemoglobin properties.

It is our purpose to review recent experiments on haemoglobin in order to discuss them in terms of the two state model of cooperativity. Excellent previous reviews are available of the chemistry of haemoglobin (Antonini & Brunori, 1971; Gibson, 1959 b ) which are referred to when possible. The plethora of data necessitates that a selection must be made in a review. An intentionally wide range of experiments is selected to exhibit

High Resolution Nuclear Magnetic Resonance Study of the Histidine-Aspartate Hydrogen Bond in Chymotrypsin and Chymotrypsinogen

Abstract A high resolution proton nuclear magnetic resonance study of chymotrypsin A δ and Chymotrypsinogen A in water has shown a single resonance at very low magnetic fields (− 18 to − 15 p.p.m. relative to dimethyl-silapentane-sulfonate). From its pH dependence (p K = 7·2) and response to chemical modification the resonance has been assigned to the hydrogen-bonded proton between His-57 and Asp-102.

Excited states of nucleotides and singlet energy transfer in polynucleotides.

We have measured the absorption, fluorescence, and phosphorescence spectra of nucleotides at 77°K. The order of decreasing energies is A, U, T, G, C for the singlet, and U−, C, G, T−, A, T for the triplet. Besides the quantum yields of fluorescence and phosphorescence, we also determined the yield for intersystem crossing to the triplet, using the amplitude of the Δm=2 triplet ESR signal. We find internal conversion occurring from both the singlet and triplet states. From the excitation energies in the neutral and ionized molecules, the stability of the ionizable proton in the excited states can be compared to that in the ground state, and the results are applied to the question of proton transfer from T to A in DNA.From a discussion of the various cases of intermolecular energy transfer for singlets, and considering the broadband spectra of the nucleotides, it is shown that Forsters ``very weak case is applicable. We emphasize that two subcases must be distinguished, depending on whether transfer occu...

Triplet State of DNA

Additional proof has been obtained for the fact that the triplet state in DNA and poly dAT resides at the thymine residue by comparing the intensities of the triplet ESR and phosphorescence signals from DNAs from various sources having different (A+T)/(G+C) ratios. The TMP triplet which is not populated from the exited singlet state in dilute neutral solutions can be populated by having the TMP present in high concentrations or through energy transfer from the triplet state of acetone or acetophenone. The triplet state produced in this manner has been characterized by its Δm=2 ESR line, its phosphorescence spectrum, its decay time, and the effect of deuterium substitution on the latter. In all these respects the DNA and poly dAT triplet resembles the neutral TMP triplet (T) more closely than it resembles the ionized (pH 12) TMP triplet (T−). A determination of the acidity constants of the excited states of A and T gives strong evidence against single‐proton transfer from the excited singlet or triplet st...

High Resolution Nuclear Magnetic Resonance Studies of the Active Site of Chymotrypsin I. The Hydrogen Bonded Protons of the "Charge Relay" System

Abstract High resolution proton nuclear magnetic resonance has been used to observe protons at the active site of chymotrypsin Aδ and at the same region of chymotrypsinogen A. A single resonance with the intensity of one proton is located in the low field region of the nuclear magnetic resonance spectrum. This resonance is observed in H2O solutions but not in 2H2O. On going from low to high pH the resonance titrates upfield 3 parts per million in both proteins and has a pK of 7.5. The titration can be prevented by alkylating His57 with either of two active site directed chloromethyl ketones. Using these data the proton resonance has been assigned to a proton in a hydrogen bond between His57 and Asp102. Further confirmation of this assignment lies in the observation of a similar resonance in this same low field region of the nuclear magnetic resonance spectrum of trypsin, trypsinogen, subtilisin BPN′ and α-lytic protease all of which have the Asp-His-Ser triad at their active sites. This proton resonance in chymotrypsin Aδ was used as a probe to monitor the charge state of the active site upon formation of a stable acyl-enzyme analogue N2(N-acetylalanyl)-N1benzoylcarbazoyl-chymotrypsin Aδ. In this derivative the His-Asp proton resonance titrates from the same low pH end point as in the native enzyme, −18 parts per million, to a new high pH end point of −14.4 parts per million (versus −15.0 parts per million in the native enzyme). The difference of 0.6 parts per million in the high pH end points between the native and acyl enzyme is interpreted as supporting the suggestion that a hydrogen bond exists between Ser195 and His57 in the native enzyme and zymogen. We conclude from these studies that the charge relay system from Asp102 across His57 to Ser195 is intact in chymotrypsin Aδ and chymotrypsinogen A, and that, in the native enzyme, it slightly polarizes Ser195.

Extended X-ray absorption fine structure determination of iron nitrogen distances in haemoglobin

EXAFS spectra have been obtained of oxy and deoxy complexes of haemoglobin and of the ‘picket fence’ porphyrin, using synchrotron radiation as a source of X rays. The fluorescence data were Fourier filtered to obtain distances to the first shell and corrections applied to remove contributions from the axial ligands. In this way, the iron to porphinato nitrogen distances were determined to be 1.98±0.01 Å for both oxygenated complexes and 2.055±0.01 Å for both deoxy forms.

An allosteric model of hemoglobin: I, kinetics

Abstract The allosteric model of Monod, Wyman & Changeux (1965 ; the Monod model) has been used to analyze the kinetics of ligand binding to mammalian hemoglobins. Combination velocity constants and dissociation velocity constants for the T and R forms were evaluated from the early measurements by Roughton & Gibson of the oxygen and carbon monoxide binding to sheep hemoglobin at pH 9.1 and more recent measurements of oxygen binding to human hemoglobin at pH 7. The difference in ligand affinities between the T and R forms, which is a factor of several hundred, is taken up almost equally by the combination and dissociation rates. The allosteric model is shown to be particularly suitable for explaining the flash photodissociation of carboxyhemoglobin. The monotonic decrease of the fraction of quickly reacting form with increasing flash intensity shown by Antonini, Chiancone & Brunori (1967) and the flow-flash experiments of Gibson & Parkhurst (1968) are both in excellent agreement with the calculations on the Monod model, with no adjustable parameters except for the fit to the equilibrium curves. In flow experiments, both the forward progess curves and the reverse dissociation curves after mixing with dithionite are very well described by the Monod model. Finally, Gibsons recent flow experiments on human hemoglobin at pH 7 have been shown to be consistent with the parameters of the Monod model which can describe the other kinetic experiments. From all this, we conclude that the kinetic experiments discussed can be fitted by the simple Monod allosteric model to well within the range of experimental errors, and that the kinetic data now available are not inconsistent with this model.

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.