Department of Biochemistry, Bose Institute, P-1/12 CIT Scheme VIIM, Calcutta 700 054, India
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Abstract |
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Keywords: conformation/protein engineering/protein structural comparison/secondary structure/structural plot
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Introduction |
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We have recently shown (Chakrabarti and Pal, 1998) the interdependence of the main- and side-chain torsion angles (
,
and
1), and there is also a need for better understanding of the role of the side-chain in the packing and stability of the protein fold. As such, an unambiguous representation of
1 is as important as the
chain trajectory, and a plot discussed here meets this requirement.
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Materials and methods |
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The y-axis of the plot is divided into four major bands corresponding to the four regions of the Ramachandran map containing non-overlapping clusters of ,
points (Chakrabarti and Pal, 1998
) (Table I
). Each major region is further subdivided into four groups based on
1 (Table II
).
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Multi-chain files can easily be processed as one specifies the range to be plotted by providing the serial numbers of the residue records in the `.rin' file. Any sequence length can be handled by the program. However, for a long chain, if the labels along the x-axis become cluttered, one can split it into smaller segments to be plotted separately (a maximum of nine allowed on a single page). If one is interested in the trace only, it is also possible to put labels at an interval on the x-axis.
For overlaying the second (and subsequent) plots (drawn with broken lines with varying gap widths) the required number of `.rin' files are read. Polypeptide chains with similar structures but different sequences (and residue numbers) can be plotted together, but the prior information on sequence alignment and gap positions have to be provided.
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Results and discussion |
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For clarity, instead of plotting the three torsional angles, we have identified each residue by the well-defined regions in the three-dimensional conformational space in which it occurs. So the ,
values, depending on the location on the Ramachandran map, are replaced by a single label A, B, L or R, corresponding to four regions in the Ramachandran mapthe first three are named after the prominent secondary structural element each encompasses (A,
-helix; B, ß-strand; L, left-handed
-helix; R, the remaining region). Likewise, the side-chain conformation is shown not by the actual
1 value, but by three idealized states, gauche (g+ and g) and trans (t); Gly and Ala, with no
1, are shown against `x' along the y-axis. Besides the conformational data, the secondary structural features of residues, disulfide linkages are also given. If needed, additional panels can be inserted to show residues involved in function, subunit association or substrate binding. A few representative plots are presented in Figures 13
.
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The identification of a stretch of backbone with a right- or left-handed -helical and ß-strand conformation is very easy as these appear in separate bands. However, in going from one residue to the next, if the change in conformation does not involve a shift from one to another in the four distinct regions of the Ramachandran map, it does not show up in the plot. As a result, it cannot distinguish between
- and 310-helices or parallel and antiparallel ß-sheets which can be identified only if the
,
values are plotted, but even then, as the differences are small the patterns in the plot would be nearly identical (McClain and Erickson, 1995
). Of the two widely occurring ß-turns, the type II turn with the two central residues having
,
combinations of [(60,120) and (80,0)] involving a B
L change is easily recognizable, but not the type I turn [(60,30) and (90,0), A
A change]. Such ambiguities can be resolved from the secondary structural information provided below the residue names. The plot can very elegantly display a change in conformation from one region of the Ramachandran map to another, especially when the sequence of residues do not constitute any regular secondary structure. Likewise, the occurrence in region R, which is not very common for a non-Gly residue, is clearly shown, and the context in the primary and secondary structures in which such a residue is located can be analysed from the plot.
Side-chain conformation
One of the motivations for designing the plot was to see if there is any pattern in the side-chain conformation as one moves along the polypeptide chaina matter which has not received adequate attention in the literature. For example, considering the four distinct possibilities (no 1 and the three conformational states of
1) in a given region, one may ask the question if a residue with no
1 occurs at any specific position, or for other residues, if any combination of the conformational states is preferred in adjacent positions or with a fixed interval.
Structural comparison
The plot provides an easy visual comparison of protein structures, as shown in Figure 1, for the two crystal forms of hen egg white lysozyme. There are changes not only in the side-chain conformation of a few residues, but also in two stretches of the main chain (residues 7374 and 103104). The changes in the side-chain conformation usually get overlooked as most of the publications report the root mean square deviation calculated using backbone atoms only. However, we have recently shown that the residues having multiple side-chain conformation usually have their main-chain torsion angles restricted (Chakrabarti and Pal, 1998
). In another example, Figure 2
compares the structures of metallothionein (Braun et al., 1992
) obtained using X-ray and NMR methods, and indicates that the non-ligand residues have significant conformational differences.
We have been interested in the structure and dynamics around the cis peptide linkages (Pal and Chakrabarti, unpublished work) and came across a study on a Lys116Gly mutation in staphylococcal nuclease (Hodel et al., 1993
). The peptide bond between residues Lys116 and Pro117 is cis in the wild-type structure but becomes trans in the mutant. The structural changes were reported to be limited not only in the region of mutation (111119) but also in regions further away (4451). The consideration of side-chain conformation in the plot indicates that there are many more alterations in the structure. The plot can thus highlight changes accompanying protein engineering experiments.
Since the plot is easy to visualize, additional parameters can be displayed for other applications. One can use this plot to highlight the disordered regions of a molecule to find out if there is any link with the structure and the sequence they are embedded in. NMR models which give an ensemble of structures can be plotted to visualize variation along the sequence. Flexibility associated with the surface accessibility may also be assessed from these plots.
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Conclusion |
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The software is available for academic use from the authors, and the file (pub/pinak/confplot/confplot.tar.gz) can be downloaded from boseinst.ernet.in.
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Acknowledgments |
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Notes |
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References |
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Bernstein,F.C., Koetzle,T.F., Williams,G.J.B., Meyer,E.F.,Jr., Brice,M.D., Rodgers,J.R., Kennard,O., Shimanouchi,T. and Tasumi,M. (1977) J. Mol. Biol., 112, 535542.[ISI][Medline]
Braun,W., Vasak,M., Robbins,A.H., Stout,C.D., Wagner,G., Kägi,J.H.R. and Wüthrich,K. (1992) Proc. Natl. Acad. Sci. USA, 89, 1012410128.[Abstract]
Chakrabarti,P. and Pal,D. (1998) Protein Engng, 11, 631647.[Abstract]
Hodel,A., Kautz,R.A., Jacobs,M.D. and Fox,R.O. (1993) Protein Sci., 2, 838850.
Hynes,T.R. and Fox,R.O. (1991) Proteins Struct. Funct. Genet., 10, 92105.[ISI][Medline]
Laskowski,R.A., MacArthur,M.W., Moss,D.S. and Thornton,J.M. (1993) J. Appl. Crystallogr., 26, 283291.[ISI]
McClain,R.D. and Erickson,B.W. (1995) Int. J. Pept. Prot. Res., 45, 272281.[ISI]
Ramachandran,G.N., Ramakrishnan,C. and Sasisekharan,V. (1963) J. Mol. Biol., 7, 9599.[ISI][Medline]
Ramanadham,M., Seiker,L.C. and Jensen,L.H. (1990) Acta. Crystallogr., B46, 6369.[ISI]
Richardson,J.S. (1981) Adv. Prot. Chem., 34, 167339.[Medline]
Sheriff,S., Hendrickson,W.A. and Smith,J.L. (1987) J. Mol. Biol., 197, 273296.[ISI][Medline]
Srinivasan,R., Balasubramanian,R. and Rajan,S.S. (1975) J. Mol. Biol., 98, 739747.[ISI][Medline]
Srinivasan,A.R. and Yathindra,N. (1978) Biopolymers, 17, 15951600.[ISI]
Venkatachalam,C.M. (1968) Biopolymers, 6, 14251436.[ISI][Medline]
Received February 19, 1999; revised April 14, 1999; accepted April 14, 1999.