Structures using X-ray diffraction data collected to 1.5-A resolution have been determined for the protein ribonuclease-A at nine different temperatures ranging from 98 to 320 K. It is determined that the protein molecule expands slightly (0.4% per 100 K) with increasing temperature and that this expansion is linear. The expansion is due primarily to subtle repacking of the molecule, with exposed and mobile loop regions exhibiting the largest movements. Individual atomic Debye-Waller factors exhibit predominantly biphasic behavior, with a small positive slope at low temperatures and a larger positive slope at higher temperatures. The break in this curve occurs at a characteristic temperature of 180-200 K, perhaps indicative of fundamental changes in the dynamical structure of the surrounding protein solvent. The distribution of protein Debye-Waller factors is observed to broaden as well as shift to higher values as the temperature is increased.

Members of the transferrin family of proteins are involved in Fe3+ transport (serum transferrins) and are also believed to possess antimicrobial activity (ovotransferrins and lactoferrins). The structure of the monoferric N-terminal half-molecule of hen ovotransferrin, reported here at 2.3-A resolution, reveals an unusual interdomain interaction formed between the side-chain NZ atoms of Lys 209 and Lys 301, which are 2.3 A apart. This strong interaction appears to be an example of a low-barrier hydrogen bond between the two lysine NZ atoms, both of which are also involved in a hydrogen-bonding interaction with the aromatic ring of a tyrosine residue. Crystals of the protein were grown at pH 5.9, which is well below the usual pKa approximately 10 for a lysine side chain. We suggest that the pKa of either one or both of these residues lies below the pH of the structure determination and is, therefore, not positively charged. This finding may serve to explain, on a molecular basis, the pH dependence of transferrin Fe3+ release. We propose that uptake of the Fe(3+)-transferrin complex into an acidic endosome (viz., pH approximately 5.0) via receptor-mediated endocytosis will result in the protonation of both lysine residues. The close proximity of the two resulting positive charges, and their location on opposite domains of the N-lobe, might well be the driving force that opens the two domains of the protein, exposing the Fe3+ ion and facilitating its release.(ABSTRACT TRUNCATED AT 250 WORDS)

X-ray diffraction data from monoclinic crystals of yeast tRNAPhe soaked in dilute lead(II) acetate solutions at pH 5.0 and at pH 7.4 have been collected to a resolution of 3 A, and the Pb(II) binding sites have been obtained by difference Fourier analyses. The same three Pb(II) binding sites are observed at both of these pH values. At pH 7.4 an extra peak of negative electron density appears on the difference map close to one of the Pb(II) binding sites and at the position of phosphate-18, indicating cleavage of the sugar-phosphate-chain between residues D-17 and G-18 of the tRNAPhe molecule in this derivative. Chain scission does not occur to any observable extent in the structure at pH 5.0, and we have, therefore, a picture of the reactants (at pH 5.0) and products (at pH 7.4) of this cleavage reaction. Polyacrylamide gel electrophoresis as well as sequencing experiments confirms the cleavage of the tRNAPhe molecule into one-fourth and three-fourth fragments, with the shorter fragment consisting essentially of residues G-1 through D-17 while the larger fragment contains residues G-18 through A-76. End-group analyses suggest a ribose cyclic 2',3'-phosphate at D-17 of the one-fourth fragment with a 5'-OH at G-18 of the three-fourth fragment. Cleavage of the tRNAPhe molecule does not occur in the absence of Pb(II), and the proximity of one of these metal ions to the cleavage site strongly implicates this metal ion in the cleavage reaction. Consideration of several possible mechanisms for the reaction, taking into account the biochemical and crystallographic facts presented above, suggests that the cleavage involves removal of the proton from the 2'-OH of ribose-17 by a Pb(II)-bound hydroxyl group. Subsequent nucleophilic attack of the resulting 2'-O- on the phosphorus atom of phosphate-18, presumably through a pentacoordinate phosphorus cyclic intermediate (as in the action of pancreatic ribonuclease A), results in chain scission. It cannot be decided whether the displacement, within the pentacoordinate intermediate, proceeds via an in-line or adjacent pathway, but an exploration of the likelihood of either pathway is presented. Strand cleavage at the particular site occurs fortuitously because the aquo Pb(II) ion binds at the correct distance and presumably in such a manner as to present a hydroxyl group in the correct orientation to effect the proton abstraction.(ABSTRACT TRUNCATED AT 400 WORDS)

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Pb(II) is extremely efficient at depolymerizing RNA and studies on tRNAs have shown that site-specific cleavages in these molecules can be brought about by the action of Pb(II). We have observed, by difference Fourier analysis, sugar-phosphate strand scission between residues 17 and 18 in crystals of yeast tRNAPhe soaked in dilute Pb(II) solution at pH 7.4. We have also deduced the structure of the Pb(II)-tRNAPhe derivative at pH 5.0 where this cleavage reaction is considerably slower and report that, in this structure, the sugar-phosphate backbone remains intact. We have, therefore, a picture of the reactants (at pH 5.0) and products (at pH 7.4) of this cleavage reaction. From this crystallographic study, and associated biochemical work, we have formulated a possible mechanism for the cleavage reaction and also present here some general ideas on the action of metal ions on nucleic acids.

1423actual dissociation (AD2 -AD + D) in which the leaving molecule carries away the excess energy. SummaryThe emission properties of jet-cooled adducts between ethers and some aromatic hydrocarbons may be accounted for by assuming an excited state that is a superposition of a van der Waals locally excited (LE) state and a charge-transfer (CT) state. The direct optical transition from the ground state to the C T state is forbidden by either symmetry or Franck-Condon considerations. Thus, the excitation spectrum is dominated by the LE state, and emission from the CT state has a longer decay time. The C T character of the excited state is more prominent in anthracene and its derivatives than in perylene. This is particularly so when a hydrogen atom is bound to a C-9 or C-IO carbon. It is therefore proposed that this atom may serve as a bridge in transmitting the charge from the oxygen lone pair orbital of the ether. Geometric factors appear to be decisive in determining the interaction as shown by the fact that tetrahydrofuran, in which the oxygen atom is constrained to be away from the 9-or 10-position, does not show exciplex-type emission with anthracene. Abstract:The reaction between W(C-t-Bu)(dme)CI, and ArNH(TMS) (Ar = 2,6-C,H3-i-Pr2) yields W(C-t-Bu)(NHAr)(dme)CI,.

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