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This study addresses a void in the existing literature on the amide-15 N chemical shift anisotropy (CSA) tensor of peptides: a systematic investigation of how the tensor varies in different peptides. Amide-15 N CSA tensors for several dipeptides are obtained using quantum chemical calculations, as well as for a series of model Ala-X and X-Ala sequences in both α-helical and β-sheet conformations (where X is one of the naturally occurring amino acids). The calculated values show a significant variation in both isolated and extended peptide structures. Hydrogen bonding at both the carbonyl group and the N-H bond of the peptide plane is shown to affect the principal values of the tensor. Calculations on model peptides indicate that the amide-15 N CSA tensor is dependent on atoms located within a distance of five bonds. Consequently, the tensor of a given peptide residue is unaffected by residues other than those adjacent to it, which implies that the amide-15 N CSA tensor should be considered in the context of tripeptide sequences. This further suggests that the amide-15 N CSA tensor of the second residue of a given tripeptide sequence may be extrapolated to the same sequence in any other polypeptide or protein, given the same backbone conformation and intermolecular environment. These conclusions will facilitate future NMR structural studies of proteins.

Most of the small ribozymes, including those that have been investigated as potential therapeutic agents, appear to be rather poor catalysts. These RNAs use an internal phosphoester transfer mechanism to catalyze site-specific RNA cleavage with apparent cleavage rate constants typically <2 min ؊1 . We have identified variants of one of these, the Neurospora Varkud satellite ribozyme, that self-cleaves with experimentally measured apparent rate constants of up to 10 s ؊1 (600 min ؊1 ), Ϸ2 orders of magnitude faster than any previously characterized self-cleaving RNA. We describe structural features of the cleavage site loop and an adjacent helix that affect the apparent rate constants for cleavage and ligation and the equilibrium between them. These data show that the phosphoester transfer ribozymes can catalyze reactions with rate constants much larger than previously appreciated and in the range of those of protein enzymes that perform similar reactions. S equence-or structure-specific cleavage of RNA phosphodiester bonds by many protein enzymes is quite rapid: for example, ribonuclease III cleaves its target RNA structure with an apparent rate constant (k obs ) of 6.4 s Ϫ1, and RNaseA can cleave its preferred dinucleotide sequence even faster, from 15.2 to 675 s Ϫ1 , depending on the source of the enzyme (1, 2). Site-specific hydrolytic cleavage of RNA by the RNA subunit of Bacillus RNaseP or the Tetrahymena self-splicing group I intron has been observed (3) or calculated (4) to be fast, in the range of 6 s Ϫ1 . A rate constant of Ϸ10 s Ϫ1was measured for a ligase ribozyme obtained by in vitro selection to catalyze the attack of a 3Ј hydroxyl on a 5Ј triphosphate (5).In contrast, most ribozymes appear to be rather poor catalysts. The ''small ribozymes,'' comprising the naturally occurring hammerhead, hairpin, hepatitis delta virus, and Neurospora Varkud satellite (VS) ribozymes, catalyze a transesterification reaction, yielding cleavage products with 2Ј3Ј cyclic phosphate and 5Ј hydroxyl termini like those produced by many protein ribonucleases. The vast majority of ribozymes selected in vitro to cleave RNA phosphodiester bonds also use this same phosphoester transfer chemistry and, like their natural counterparts, have cleavage rate constants of ϽϷ2 min Ϫ1 (0.033 s Ϫ1 ) (6, 7). A variety of enzymological considerations that affect ribozyme reaction rates have been discussed (8), and it has been recently proposed that chemical principles may limit the rates of certain small ribozymes (9, 10).The VS ribozyme is found in RNA transcripts of a plasmid in the mitochondria of certain natural isolates of the fungus Neurospora (11). It catalyzes site-specific cleavage and ligation reactions, similar to those performed by hammerhead, hairpin, and hepatitis delta virus ribozymes that are involved in the replication of the RNAs that contain the ribozyme (reviewed in refs. 12 and 13). Cleavage in VS RNA occurs after nucleotide G620 in an internal loop between helices Ia and Ib (Fig. 1B) (14). Biophysical, crosslinking, mu...

This study reports magnitudes and the orientation of the (13)C(alpha) chemical shift anisotropy (CSA) tensors of peptides obtained using quantum chemical calculations. The dependency of the CSA tensor parameters on the energy optimization of hydrogen atom positions and hydrogen bonding effects and the use of zwitterionic peptides in the calculations are examined. Our results indicate that the energy optimization of the hydrogen atom positions in crystal structures is necessary to obtain accurate CSA tensors. The inclusion of intermolecular effects such as hydrogen bonding in the calculations provided better agreement between the calculated and experimental values; however, the use of zwitterionic peptides in calculations, with or without the inclusion of hydrogen bonding, did not improve the results. In addition, our calculated values are in good agreement with tensor values obtained from solid-state NMR experiments on glycine-containing tripeptides. In the case of peptides containing an aromatic residue, calculations on an isolated peptide yielded more accurate isotropic shift values than the calculations on extended structures of the peptide. The calculations also suggested that the presence of an aromatic ring in the extended crystal peptide structure influences the magnitude of the delta(22) which the present level of ab initio calculations are unable to reproduce.

The cleavage site of the Neurospora VS ribozyme is located in an internal loop in a hairpin called stem-loop I. Stem-loop I undergoes a cation-dependent structural change to adopt a conformation, termed shifted, that is required for activity. Using site-directed mutagenesis and kinetic analyses, we show here that the insertion of a single-stranded linker between stem-loop I and the rest of the ribozyme increases the observed self-cleavage rate constant by 2 orders of magnitude without affecting the Mg(2+) requirement of the reaction. A distinct set of mutations that favors the formation of the shifted conformation of stem-loop I decreases the Mg(2+) requirement by an order of magnitude with little or no effect on the observed cleavage rate under standard reaction conditions. Similar trends were seen in reactions that contained Li(+) instead of Mg(2+). Mutants with lower ionic requirements also exhibited increased thermostability, providing evidence that the shifted conformation of stem-loop I favors the formation of the active conformation of the RNA. In natural, multimeric VS RNA, where a given ribozyme core is flanked by one copy of stem-loop I immediately upstream and another copy 0.7 kb downstream, cleavage at the downstream site is strongly preferred, providing evidence that separation of stem-loop I from the ribozyme core reflects the naturally evolved organization of the RNA.