1 Centro di Studio di Biocristallografia CNR and Dipartimento di Chimica, Università di Napoli `Federico II', Via Mezzocannone 4, I-80134 Naples and 2 CEINGE, Biotecnologie Avanzate, Naples, Italy
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Abstract |
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Keywords: amide resonance/crystallography/peptide bond/protein structure/ultrahigh resolution
Abbreviations: Beq, equivalent B-factor CSD, Cambridge Structural Database N67isoD-RNase A, ribonuclease A containing an isoaspartyl residue at position 67 PDB, Protein Data Bank PPI, peptidylprolyl cistrans isomerase
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Introduction |
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In this paper, we consider a further aspect of the peptide group, i.e. the correlation between CO and CN bond lengths as expected from the classical Pauling resonance model. In fact, it posits the contribution of the polar form, >N+=CO, to the peptide structure, with a partial double bond character for the CN bond, and it implies an increase in the C=O bond order when the CN bond order decreases. The simple resonance model for the amide structure has been debated recently (Wiberg and Breneman, 1992; Fogarasi and Szalay, 1997
) since some authors have suggested a more complex picture which, in addition to the
-orbital overlap, emphasizes the role of
-bond polarization and of the Coulombic interactions between covalently linked atoms (Milner-White, 1997, and references cited therein).
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Results and discussion |
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In the PDB, eight protein structures were selected when considering only anisotropically refined models containing >50 residues and determined at a resolution of better than 0.95 Å (see Table I for references). Again, peptide groups were selected on the basis of the equivalent B-factors of backbone atoms. In five structures, the negative correlation is statistically significant and the linear fitted equations appear to be rather similar (Table I
). For two structures the regression line shows a negative slope, although the correlation is less significant. In the remaining structure the value of the slope is close to zero, an indication that there is no correlation. Figure 1C
illustrates the results of the regression analysis of the five protein structures and for A and B molecules of N67isoD-RNase A. The data suggest that the correlated variations in peptide geometry are real, even though sometimes they may be hidden by the stereochemical restraints used in the refinement.
Although already traced in small molecules, the negative correlation between protein CO and CN bond distances represents a new and important result. These geometry changes in small peptides are mostly due to simple crystal field effects, whereas in proteins they can be produced by intramolecular interactions. Preliminary attempts to connect the bond length changes with specific structural motifs or hydrogen bonding environments did not produce consistent results. As an example, the examination of the CO bond lengths in the better defined regions of -helices of the N67isoD-RNase A structure showed that, on average, the CO distance is shorter (~0.01 Å) when the oxygen is not involved in the hydrogen bond typical of
-helices. The difference in bond lengths is, however, not statistically meaningful. The failure to detect the origins of the CN/CO distance variations can be ascribed to the limited database of ultrahigh-resolution structures and also to the high complexity of a macromolecular framework. In proteins, almost every oxygen and nitrogen atom of peptide groups forms hydrogen bonds and it is difficult to evaluate the strength of the various interactions. A rigorous analysis should take into account not only the number and the stereochemical features of hydrogen bonds, but also the occupancies and B-factors of the atoms involved, the long-range interactions, the role played by the solvent and the placement of the peptide bond in the interior or on the surface of the protein. In particular, an accurate definition of the local electrostatic potential near each peptide group is required.
The results presented here are consistent with Pauling's classical model of amide resonance and at the same time contribute to changing the traditional view of peptide groups usually considered as fairly rigid structural units. In proteins, peptide bonds actually undergo subtle changes in the electronic distribution, which are then reflected in geometry differences.
The importance of these subtle electronic variations has been highlighted by studies on peptidylprolyl cistrans isomerases (PPI) (VanDuyne et al., 1991) which catalyse the interconversion of cis and trans isomers of peptide bonds, a process which can often have a rate-limiting role in protein folding. It has been suggested that in PPI the catalytic action occurs by burying the proline amide bond in a hydrophobic cavity. This process destabilizes amide resonance forms in which the oxygen is negatively charged, thus favouring the resonance structures in which the amide carbonyl is more ketone-like and consequently lowering the rotational barrier around the CN bond (Eberhardt et al., 1992; Harrison and Stein, 1992
). The present findings give further support to the hypothesized mechanism by showing a certain degree of flexibility of the peptide linkage, whose electronic state and geometry can be influenced by the environment.
In conclusion, the analysis of very high-resolution protein structures can reveal subtle information about local electronic features of proteins, which may be critical to folding, function or ligand binding. A deeper understanding of the structural properties may be of importance for further progress in structure prediction methods. On the other hand, the accuracy of these methods could be evaluated by their ability to reproduce the fine details found in very high-resolution protein structures.
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Materials and methods |
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An estimate of the positional uncertainties has been derived at the end of the refinement by a blocked matrix inversion (Esposito et al., 2000a).
The CSD database was searched for the fragment, CHC(=O)NCH, belonging to peptide sequences and not being N-acetyl or N-methylamide terminal units. Metal-containing complexes were excluded and also data from cyclic, disordered or D-amino acids containing peptides. Furthermore, structure determinations resulting in an R value >0.07 were rejected.
To evaluate the validity of the correlation coefficients between CO and CN distances, the so-called null hypothesis, i.e. the hypothesis that the variables are not correlated, was tested by using Student's t distribution. The statistical test yields a p-value (reported in Table I) which represents the probability that random sampling would result in a correlation coefficient as far from zero as observed in our data set, under the hypothesis that there is no correlation between the two variables; p-values <0.05 allow one to reject the null hypothesis at the 95% confidence level.
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Notes |
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Acknowledgments |
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References |
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Received August 10, 2000; revised October 12, 2000; accepted October 12, 2000.