PDBsum entry 261l

Hydrolase

PDB id: 261l

Contents

Protein chain

173 a.a. *

Waters ×43

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Structural characterization of an engineered tandem repeat contrasts the importance of context and sequence in protein folding.

Authors

M.Sagermann, W.A.Baase, B.W.Matthews.

Ref.

Proc Natl Acad Sci U S A, 1999, 96, 6078-6083. [DOI no: 10.1073/pnas.96.11.6078]

PubMed id

10339544

Abstract

To test a different approach to understanding the relationship between the sequence of part of a protein and its conformation in the overall folded structure, the amino acid sequence corresponding to an alpha-helix of T4 lysozyme was duplicated in tandem. The presence of such a sequence repeat provides the protein with "choices" during folding. The mutant protein folds with almost wild-type stability, is active, and crystallizes in two different space groups, one isomorphous with wild type and the other with two molecules in the asymmetric unit. The fold of the mutant is essentially the same in all cases, showing that the inserted segment has a well-defined structure. More than half of the inserted residues are themselves helical and extend the helix present in the wild-type protein. Participation of additional duplicated residues in this helix would have required major disruption of the parent structure. The results clearly show that the residues within the duplicated sequence tend to maintain a helical conformation even though the packing interactions with the remainder of the protein are different from those of the original helix. It supports the hypothesis that the structures of individual alpha-helices are determined predominantly by the nature of the amino acids within the helix, rather than the structural environment provided by the rest of the protein.

Figure 2.
Fig. 2. (A) Initial electron density showing the overall conformation of the duplicated sequence, as seen in space group P3[2]21. The WT* structure, omitting residues 36-42 (shown as a ribbon drawing) was subject to 10 cycles of rigid-body refinement in the mutant lysozyme cell. The calculated phases and structure factors, F[c], were used to calculate a map with amplitudes (F[mutant] F[c]) at 3.0-Å resolution. The density in the vicinity of the deleted residues, contoured at 2.5 , is shown. (B) Electron density after refinement of the inserted region in space group P3[2]21. Coefficients are (2F[o]-F[c]). The structure factors, F[c], and phases were calculated from the refined model including the inserted region. The resolution is 2.5 Å, and the map is contoured at 1.0 . (C) Superposition of the overall structure of the duplication mutant in space group P3[2]21 (blue bonds) on WT* lysozyme (green bonds). The inserted region in the mutant structure is highlighted in yellow.

Figure 3.
Fig. 3. (A) Map showing the initial electron density for the inserted region of molecule A in space group P2[1]. Amplitudes are (2F[o]-F[c]) weighted by REFMAC (15) where the structure factors, F[c], and phases were calculated from the refined model including the inserted region. The map was calculated at 2.5-Å resolution and contoured at 1.0 . The density in the vicinity of residues 40i-43i is not well defined and could not be fit by a well-defined model. (B) Electron density for molecule B of crystal form P2[1]. This map was calculated with the same coefficients, contouring, and resolution as in A. (C) Superposition of the C trace of the two copies of mutant L20 in crystal form P2[1] (molecule A, blue; molecule B, mauve) and wild-type T4 lysozyme (green). The sequence of the insert is highlighted in yellow for molecule A and in orange for molecule B. The structural rearrangements of loop 18-25 in molecule B are clearly visible. The superpositions were based on the -carbon atoms of residues 51-80 within the amino-terminal domain. Because of slight changes in the hinge-bending angle the C-terminal domains appear out of register although the respective structures within these regions are very similar.

Secondary reference #1

Title

Protein structural plasticity exemplified by insertion and deletion mutants in t4 lysozyme.

Authors

I.R.Vetter, W.A.Baase, D.W.Heinz, J.P.Xiong, S.Snow, B.W.Matthews.

Ref.

Protein Sci, 1996, 5, 2399-2415. [DOI no: 10.1002/pro.5560051203]

PubMed id

8976549

Figure 3.
Fig. 3. Conrmued.

Figure 12.
Fig. 12. Diagrammaticanalysis of heinterfacebetweenthe``cap''formed by esidues 14-142andthe remainder of thecarboxy- terminaldomain in WT*lysozyme.Therepresentationismadeupof two panels. A: Summarzesalltheinteractionsthataregenerated by atomsthatformoneside fthe interface (residues 1 4-142). : howstheinteractionsthatareformed by allthe atoms ontheother side ofthe interface.Blackdotsshowvan der Waals contactsbetweenpairs of atomswhosecentersarewithin4.0 A. Residuestha participate in hesecontacts re umbered.Atomsthatparticipateinhydrogenbonds across the interface are indicated byred spheres, saltbridgesareindicated by purplespheres,andothernonpolarcontactsareshown by greenspheres.Greencontourlinesshowthe separation across theinterfacebetweenthevanderWaalssurfaceswithcontourscorrespondintodecreasingseparations in steps of 0.1 A. In general,thblackdots(nonpolar contacts) and oloredspheres (polar contacts) tendtooccurat or neartheminima of the green contours becausethesecorrespondtotheclosestapproaches across theinterface.Thedottedblacklinedefinstheregion of the interfacefromwhichsolvent is excluded lineisdefinedasthepath of contactthatistracedout by a atermoleculeatthesurface ofthe proteinthat just touchesboth sides ofthe nterface).Theinformation is projected onto a planethatisessntiallyparallel to th contactinterface,thesameplanebeing used forFigures 12-16. : Interaction surface formedbythe ``cap''residues 114-142. The backbonetrace of theseresidesisshown in lue. B: nteraction surface inWT* lysozymethatopposesthe surface shown in A, composed of residues 83-1 12 plus 144-154 (backbone drawninblue).

The above figures are reproduced from the cited reference which is an Open Access publication published by the Protein Society

Secondary reference #2

Title

How amino-Acid insertions are allowed in an alpha-Helix of t4 lysozyme.

Authors

D.W.Heinz, W.A.Baase, F.W.Dahlquist, B.W.Matthews.

Ref.

Nature, 1993, 361, 561-564.

PubMed id

8429913