shells with SA#20%. Glu is most frequently observed in the protein shell with SA between 50 and 60%. Interestingly, both Asp and Glu do not like to be fully solvent accessible. Figure 3C shows that the distribution of Lys displays a Gaussian profile, peaking in the 40–50% SA shell, and falls off rapidly both with increasing and decreasing SA. Interestingly, the distribution of Arg is similar to the distribution of the oppositely charged Asp residue, preferring to be located in protein shells displaying solvent accessibility .0% and #60%. All four titratable residues avoid being located in the totally buried core (0% SA) of the protein, as well as in the highly solvent accessible shells. On the other hand, Cys residues prefer to be buried in proteins. Its distribution peaks in shells with SA#10%, decaying afterwards exponentially towards increasing SA shells. Cys is almost completely absent in shells with SA.80%. The secondary structural preferences for Asp, Glu, Lys, Arg and Cys are also displayed in Figure 3. Asp displays a preference for being located in coil structures in all solvent shells. Glu prefers to be located in a-helices whereas Lys and Arg display a preference for both a-helix and coil structures with a slight preference for the former. In general, for Glu, Lys and Arg the fractions of residues in coil and turn increase with solvent accessibility. For all four residues, the fraction of residues in b-strands decreases in an exponential way in protein shells with SA.50–60%. Cys prefers to be located both in a-helix or b-strands if completely buried (0% SA shell). In the protein shell with SA between 0–10%, Cys prefers to be located in coil structures, followed by a similar preference for a-helix and b-strands. When presence in shell with SA.10%, Cys prefers to be located in coil structures. Cysteine residues’ interactions Figure 4 displays the occurrence of Cys-Cys pairs as a function of spatial distance between the two cysteine residues forming a pair and the solvent accessibility of the protein shell where the pair is found. In Figure 4A the sequence distance between the Cys residues is equal to or less than 4 residues while in Figure 4B the sequence distance is larger than 4. For sequence distances #4, 926 pairs are found. The vast majority are found below 20% SA. The highest occurrence of pairs is seen for spatial distances between the two cysteines of 3.8–4.3 A˚ and 6.3–7.3 A˚. Those pairs are located in a protein shell with SA#10%. Furthermore, Cys-Cys pairs are also observed at spatial distances of 1.8–2.3 A˚, though less frequently. When observing Figure 4B we can see that 4968 Cys-Cys pairs are found. These pairs are also located in protein shells with SA#10% but the preferred spatial distance between these cysteine pairs is 1.8–2.3 A˚. Fewer pairs are observed at spatial distances of 3.8–4.3 A˚ and 6.3–7.3 A˚ . Protein structures containing Cys-Cys pairs representative of the two major peaks displayed in Figure 4A (sequence distance less than 4) have been retrieved with the ProPair program and are displayed in Figures 4C and 4D. Figure 4C shows that Cys-Cys pairs with distances peaking between 3.8–4.3 A˚ are part of a classical zinc finger motif in proteins. ProPair shows that the majority of the hits are zinc fingers. The local structure around the zinc finger in 2bx9.pdb, a protein involved in transcription regulation, is displayed. This protein has 12 cysteine-rich zinc- binding domains. Figure 4D shows that Cys-Cys pairs with distances peaking between 6.3–7.3 A˚, are part of yet another classical cluster: the iron sulfur cluster. The ProPair program shows that the majority of the Cys pairs with distances peaking between 6.3–7.3 A˚ are found in iron sulfur clusters. ProPair also finds that Cys residues with large sequence separation but spatially close to each other (peak at 1.8–2.3 A˚ in Fig. 4B) are involved in disulphide bridges. Amino Acid Pairs in Proteins In order to judge the uniqueness of the results displayed in Figure 4B, data has been compared with the Cys-Cys pair occurrences observed when using a dataset of randomized structures. It can be observed in Figure 4E that nearly all Cys- Cys pairs found in the randomized reference dataset are located in protein shells with 0,SA#10%. The majority of the pairs have inter-residue distances between 6.8 to 8.3 A˚. Pair occurrence decreases with decreasing distances. Almost no pairs are observed below ,3.8 A˚. The data displayed in Figure 4F was obtained by dividing the absolute data for Cys-Cys pairs with a sequence separation larger than 4 residues (Figure 4B) by the reference dataset data (Fig. 4E). It can be seen that the Cys-Cys pairs cluster in protein shells with 0–30% SA and that their distances lie between 1.8–2.3 A˚ as displayed (Fig. 4F). These preferences are absent in the randomized dataset (Fig. 4E). In Figure 5A is displayed a warp plot which merges information about the absolute number of pairs observed in Figure 4B with information about the ratio information displayed in Figure 4F. The significant red peak (1.8–2.3 A˚) that was observed in Figure 4F is seen to coincide with a high pair concentration at this location (indicated by the topography). This provides a statistical fundament of the observation. It can also be observed that the number of pairs found at distances of 3.8–4.3 A˚ and 0–10% SA is significant but that these pairs do not represent unique preferences (dark blue color). Interactions between acid and basic residues In Figure 5B is displayed a warp plot showing the statistically relevant pairwise interactions that Lys residues make with other amino acids residues and at which spatial distances those interactions occur. It can be observed that Glu and Asp residues are the closest preferred neighbors seen at distances peaking around 2.3–3.3 A˚ (red peaks). All other observed dark blue peaks are less statistically relevant interactions. Pairs between residues with opposite charge In Figure 6 is displayed the occurrence of pairs between residues with opposite charge as a function of the spatial distance between them and the solvent accessibility of the shell where the pair is found. The so called ‘‘Absolute’’ plots display the number of contacts found and the ‘‘Ratio’’ plots are the ratio between the data in the absolute plots and the corresponding data found in the reference dataset of randomized structures. Ratio plots are color coded: red codes for statistically relevant peak and dark blue for a statistically non-relevant peak. Figures 6A and 6B report the contacts found between 7784 Lys-Asp pairs. Figures 6C and 6D report the contacts found between 9649 Arg-Asp pairs. 9186 Lys- Glu pairs and 11944 Arg-Glu pairs were found (data present in Figure S3). Both absolute and ratio plots show that pairs of opposite charge are preferentially found at close distances between 2.3–3.3 A˚ allowing for salt bridge formation and in protein shells with 0,SA#50%. Pairs between residues with the same charge In Figures 7 and 8 are displayed the occurrences of pairs between same charge residues as a function of distance between any two charged residues and the solvent accessibility of the shell where the pair is found. As explained above, absolute and ratio plots are displayed for each pair. A total of 7010 Asp-Asp pairs, 9230 Glu-Glu pairs, 7302 Asp-Glu pairs, 3412 Lys-Lys pairs, 7574 Arg-Arg pairs, 3747 Arg-Lys pairs were analyzed. The ratio plots show that residues of the same charge prefer to be located further away from each other (pairs rarely seen at distances below 4.3 A˚) and in solvent layers with higher SA (40,SA#80%) when PLoS ONE | www.plosone.org 7 December 2011 | Volume 6 | Issue 12 | e25638