Amino Acid Pairs in Proteins Figure 4. Distribution of the observed spatial distance (A˚ ) and solvent accessibility of the protein shell where 923 Cys-Cys pairs located at a sequence distance #4 residues (4A) and at a sequence distance .4 residues (4B) are found. The intensity map is color coded (blue to red) and next to the color bar is displayed the number of pairs corresponding to each color code. Protein structures containing Cys- Cys pairs representative of the two major peaks displayed in 4A have been retrieved with the ProPair program and are displayed in 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. The Cys-Cys pair is displayed in dark blue and yellow. Two other Cys residues are displayed in cyan. Zn is displayed as a blue sphere. 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 Cys-Cys pair is displayed in dark blue and yellow. In the Fe4S4 cluster, Fe is displayed in orange and S in yellow. Figure 4E shows the distribution of the observed spatial distance (A˚ ) and solvent accessibility of the protein shell where 923 Cys-Cys pairs located at a sequence distance .4 residues are found in a randomized reference database (see Methods section). Figure 4F was obtained by dividing the absolute data for Cys-Cys pairs with a sequence separation larger than 4 residues (Fig. 4B) by the reference dataset data (Fig. 4E), this way displaying the statistically relevant peaks. doi:10.1371/journal.pone.0025638.g004 compared to oppositely charged residues (Figs. 7B, 7D, 7F, 8B and 8F), except for Arg-Arg pairs which prefer to be located in protein shells with 10,SA#30% at short spatial distances between 3.3– 3.8 A˚ (Fig. 8D, details below). Asp-Asp, Glu-Glu and Asp-Glu preferences The absolute and ratio plots do not necessarily provide the same information. Figure 7A shows that the majority of Asp-Asp pairs are found in 0,SA#10% shells peaking at the preferred distance of 6.8–7.8 A˚, with minor peaks at 4.3–4.8 A˚ and 3.3–3.8 A˚. Figure 7B on the other hand shows that the preferred spatial distances between residues in the majority of the statistically relevant pairs peak at 6.3–7.3 A˚ and 4.3–5.3 A˚, and that these pairs are preferentially seen at 40–60% SA. An interesting unexpected peak is observed at 2.3–3.3 A˚ and 0,SA#10%. Figure 7C shows that Glu-Glu pairs are found preferentially at 0,SA#10% and 40,SA#60%. However, Figure 7D shows that only the pairs found at 40,SA#70% are different from the reference set of randomized structures and therefore statistically relevant. Figure 7D also shows that the preferred distances between the Glu-Glu residues are 5.3–8.3 A˚. Figure 7E shows that the Asp-Glu pairs are preferentially found at 0,SA#10% with preferred distances at 4.3–4.8 A˚ and 6.3–8.3 A˚. However, Figure 7F shows that only the pairs found at 40,SA#70% are statistically relevant and that the preferred distances between the Asp-Glu residues are observed between 4.3–8.3 A˚. Distances below 4.3 A˚ are allowed in those protein shells. Both for Glu-Glu and Asp-Glu pairs an unexpected peak is observed at 2.3–2.8 A˚ and 0,SA#10%, as observed for Asp-Asp contacts. Lys-Lys, Arg-Arg and Arg-Lys preferences The Lys-Lys pair preferences are quite different from the Arg- Arg pair preferences, despite both residues being positively charged. Figure 8A shows that Lys-Lys pairs are preferentially observed at a spatial distance above 6.8 A˚ in protein shells with 30,SA#60%. Figure 8B shows that the statistically relevant contacts appear at distances above 6.3 A˚ and 40,SA#80%. Figure 8C shows that the Arg-Arg pairs are preferentially observed at low SA shells (0,SA#10%) and the preferred distances are 3.3–3.8 A˚ and above 6.3 A˚. Figure 8D shows that the only statistically relevant peaks occur at 10,SA#30% with a distance between residues of 3.3–3.8 A˚. Figure 8E shows that Arg-Lys pairs are seen in a wide range of SA, preferentially from 0,SA#50% with preferred inter-residue distances above 6.3 A˚ and between 4.3–4.8 A˚. Figure 8F shows that only the pairs found at 30,SA#70% are statistically relevant, especially the pairs found at 60,SA#70%. In these preferred protein shells, Arg-Lys pairs are observed at distances above 6.8 A˚. Secondary structural preferences of pairs of residues As mentioned above, the ratio plot of Lys-Asp reveals that the statistically relevant pairs are found between 2.3–3.3 A˚ and in protein shells with 10,SA#50% (Fig. 6B). In Figure 9A is displayed the secondary structural elements preferred by Lys and Asp pairs with a sequence distance of 2 and with inter-residue distances between 2.3–3.8 A˚ in protein shells with 20,SA#40%. A total of 85 pairs met the criteria. It can be seen that those Lys and Asp prefer to be located in b-sheets or in coil structures. In Figure 9A is also displayed a typical local 3D structure around a Lys-Asp pair with the characteristics depicted by the beta-beta peak: it can be seen that the Lys-Asp pair (Lys203-Asp201 displayed in yellow and blue) forms a salt bridge (3.4 A˚ distance between functional charged groups) and is part of a b-strand. This salt bridged pair is involved in a larger salt bridge network involving two additional residues (Glu 214 and Arg 221, displayed in brown and purple, respectively) located in a nearby a-helix. The distances between these residues allow for additional salt bridge formation. The depicted protein (1SC3.pdb) is a human caspase (interleukin-1 beta convertase), an enzyme that proteolytically cleaves the precursor form of the inflammatory cytokine interleukin 1-b into its active mature peptide. In Figure 9B the corresponding plot is displayed for a sequence distance of 4 between Lys and Asp. 342 pairs from the dataset met the selection criteria and were analyzed. It can be seen that Lys and Asp now prefer to be located exclusively in a-helices, forming a salt bridge that stabilizes one turn of the helix. The functional groups of Lys 115 and Asp 111 (displayed in yellow and blue, numbered according to 3CLJ.pdb) are within 2.8 A˚, suggesting a strong salt bridge. Furthermore, these residues are involved in a larger salt bridge network involving two additional residues (Asp 59 and Lys 62, displayed in brown and purple) located in a nearby a-helix. The distances between these residues allow for additional salt bridge formation (Fig. 9B). 3CLJ is an a-helical RNA polymerase II. In Figures 10A to 10C are displayed typical protein motifs involving Arg-Arg pairs that are in close proximity (3.3–3.8 A˚) in protein shells with 10–30%SA. These were the characteristics of the statistically most relevant and unexpected peak displayed in Figure 8D. Further parameters were used when searching for structures containing these pairs: Arg residues should be within a sequence distance larger than 4 and could be located in coil elements (Fig. 10A), in a-helices (Fig. 10B) or in b-strands (Fig. 10C). Figure 10A shows that the Arg-Arg pair can bind a phosphate group and at the same time be involved in a multiple salt bridge network. The distances between functional groups are displayed. The protein (2HNH.pdb) is the catalytic alpha subunit of the E. coli replicative DNA polymerase III. Figure 10B shows that nearby Arg-Arg pair (Arg936-Arg1244 displayed in yellow and blue, respectively) present in helical elements can be involved in a multiple salt bridge network involving 3 titratable residues (Asp1042, Asp1043 and Asp1240 displayed in brown) The closest distances between the functional groups of these residues (from 2.8 A˚ to 4.3 A˚) allow for salt bridge formation. The depicted protein is 202K.pdb. Figure 10C shows how nearby Arg-Arg pairs PLoS ONE | www.plosone.org 9 December 2011 | Volume 6 | Issue 12 | e25638