Both approaches demonstrate that the orientation of the Ca substituents, i.e. the dihedral angle y, modulates geometrical parameters, such as Dv and hC dihedral angles. Furthermore, similar trends can also be detected in the limited but very accurate sample of small molecule crystal structures, thus indicating that the shared overall picture emerging from the three independent analyses (survey on proteins, survey on peptides and computations) is particularly significant. We have shown that the AOs of the Ca-H(CH3)2 group and, to a lesser extent, of the terminal methyl group contribute to the amide p system. Therefore, the geometrical parameters of the amide moiety can also depend on the conformational states of these two flanking groups. It can no longer be taken for granted that the simple rules governing unsubstituted amides still hold for peptide-like systems. It can be thus misleading to focus the attention on the N-C = O moiety only. The present study shows that, not only are small planarity deviations possible, but they also lead to the stabilization of the peptide bond for most of the states of the Ramachandran space. According to our analysis it is therefore uncorrect to a priori assume that the maximum strength of the ‘amide resonance interaction’ is found for perfectly planar peptide geometries. Depending on y, the energetic costs of peptide planarity deviations are no longer symmetric (Figs. 2 and 3). If for a given value of y a negative Dv is favored, positive deviations are associated with a destabilization much larger than that expected on the ground of the simple resonance model. The similarity of the trends of hC/y and Dv/y indicates the central role of the y dihedral angle in modulating peptide planarity as well as the mutual influence among local degrees of freedom. From the methodological point of view, the results hereby reported also confirm that QM calculations on small peptide models can bring useful insights into the structural properties of large protein structure [39–43]. The observed discrepancies between the experimental trends and the theoretical ones resulting from computations in gas phase on the Ala1 model, can be overcome by the inclusion of a continuum solvent model in the calculations. These findings highlight the importance of a cor- rect handling of solute-solvent interactions to reproduce the experimental correlations between peptide bond geometrical parameters. The main biochemical implication of the present findings is that local effects play a major role in regulating peptide bond flexibility. Even though proteins assume an extremely complicated structure with a hierarchical juxtaposition of basic elements, it is noteworthy that the effects identified in extremely simplified systems (where the side chain is limited to a methyl group) are also detected in statistical analyses of protein and peptide structures containing twenty different aminoacids. Although the conformational prop- erties also depend on the size of the peptide chain [44–46] (for example, through the formation of secondary structure elements) or on the side chain type (e.g. through side chain/main chain interactions), we here highlight that very basic electronic effects are nevertheless present and operative, independently of the com- plexity of the peptide structure examined. This does not necessarily imply that long-range effects do not play any role in proteins. It is clear that, just to make an example, hydrogen bonds -with other peptide residues or with solvent molecules- involving the Oxygen and the Nitrogen lone pairs, can affect the geometry of a given residue, modulating, inter-alia, Dv or hC. However, as statistical analyses consider a large variety of peptide bonds in different contexts, long-range effects may be averaged out, allowing the emergence of the trends we highlight. Electronic Effects Governing Peptide Distortions Analogously, explicit solute-solvent interactions, whose impor- tance in determining the preferences between different secondary structures has already been highlighted [47,48], likely affect also peptide bond geometrical parameters. However, most of the residues in protein structures are not exposed to the solvent. Furthermore the impact of explicit water molecules in protein structure surveys is limited due to the averaging of a large number of possible coordination geometries. These considerations explain the agreement found between the prediction obtained by using a Continuum Model, as PCM, and the experimental results. Finally, other short-range non covalent interactions (nRp* interactions) between carbonyl groups of adjacent residues have been suggested to have an effect on protein structure and stability [46,49]. These orbital interactions could be at play in our Ala1 model and could be involved in determining the slight dependence of planarity deviations from the Q angle. The interesting problem of understanding the interplay between these interactions and those we focussed in the present study may be the subject of future investigation. The elucidation of the role of local effects is important to point out the impact of global structure on specific regions of the polypeptide chain. Indeed, the detection of departures from the trends dictated by local effects may lead to the identification of regions whose properties are strongly influenced by long-range effects. The convergence of long-range effects to endow a specific region of a protein with peculiar properties may be associated with important functional requirements. The accurate description of the interplay of peptide bond geometrical parameters here achieved also holds implications for protein structure determination, validation and prediction. Indeed, protein structure determinations strongly rely on the inclusion of a priori knowledge of stereochemical parameters [50]. Despite the impressive successes of protein crystallography, the methodological aspects related to the inclusion of these parameters in the refinement are still highly debated [51–53]. Our results support the idea that the context dependence of stereochemistry should be introduced in the refinement procedures of protein structures in order to enhance model accuracy [52,54,55]. In addition, the correlations here detected may be used as a validation tool for protein structures [26,56]. In particular, the fine modulation of peptide bond parameters is particularly suited for checking structures determined at high/ultra-high resolution, whose quality assessment may not be assured by standard validation programs that, in the current version, do not consider these aspects. Finally, the conformation-dependent variations of peptide geometry may represent a challenging benchmark for force fields developed for predictive modeling and molecular dynamics investigations. Interestingly, the inclusion of some conformation-dependent geometry [55] significantly improves the accuracy of Rosetta [57], one of the most used and powerful programs in protein structure prediction, as well as the refinement behavior of crystallographic protein structures [58]. These independent results suggest that even the conformation-dependent planarity deviations should be taken into proper account. It is important to note that, although small, the distortion effects can be cumulative, and, therefore, in a polypeptide chain composed of several hundred peptide units, small distortions can lead to substantial structural changes. Just to get a rough idea of the possible significance of these effects, let us consider the crystal structure of a deamidated derivative of ribonuclease A [59] refined at 0.87 A˚ resolution with a 6.8u standard deviation on the v angle. By artificially imposing all the trans v angles to 180u, we obtained a Ca trace that differs from the original one by about 6 A˚ (calculated on 121 atoms of chain A). PLoS ONE | www.plosone.org 7 September 2011 | Volume 6 | Issue 9 | e24533