PDBsum entry 1d5d

Hydrolase

PDB id: 1d5d

Contents

Protein chains

15 a.a. *

101 a.a. *

Ligands

SO4

Waters ×138

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Thermodynamic and structural studies of cavity formation in proteins suggest that loss of packing interactions rather than the hydrophobic effect dominates the observed energetics.

Authors

G.S.Ratnaparkhi, R.Varadarajan.

Ref.

Biochemistry, 2000, 39, 12365-12374. [DOI no: 10.1021/bi000775k]

PubMed id

11015216

Abstract

The hydrophobic effect is widely believed to be an important determinant of protein stability. However, it is difficult to obtain unambiguous experimental estimates of the contribution of the hydrophobic driving force to the overall free energy of folding. Thermodynamic and structural studies of large to small substitutions in proteins are the most direct method of measuring this contribution. We have substituted the buried residue Phe8 in RNase S with alanine, methionine, and norleucine. Binding thermodynamics and structures were characterized by titration calorimetry and crystallography, respectively. The crystal structures of the RNase S F8A, F8M, and F8Nle mutants indicate that the protein tolerates the changes without any main chain adjustments. The correlation of structural and thermodynamic parameters associated with large to small substitutions was analyzed for nine mutants of RNase S as well as 32 additional cavity-containing mutants of T4 lysozyme, human lysozyme, and barnase. Such substitutions were typically found to result in negligible changes in DeltaC(p)() and positive values of both DeltaDeltaH degrees and DeltaDeltaS of folding. Enthalpic effects were dominant, and the sign of DeltaDeltaS is the opposite of that expected from the hydrophobic effect. Values of DeltaDeltaG degrees and DeltaDeltaH degrees correlated better with changes in packing parameters such as residue depth or occluded surface than with the change in accessible surface area upon folding. These results suggest that the loss of packing interactions rather than the hydrophobic effect is a dominant contributor to the observed energetics for large to small substitutions. Hence, estimates of the magnitude of the hydrophobic driving force derived from earlier mutational studies are likely to be significantly in excess of the actual value.

Secondary reference #1

Title

X-Ray crystallographic studies of the denaturation of ribonuclease s.

Authors

G.S.Ratnaparkhi, R.Varadarajan.

Ref.

Proteins, 1999, 36, 282-294. [DOI no: 10.1002/(SICI)1097-0134(19990815)36:3<282::AID-PROT3>3.3.CO;2-6]

PubMed id

10409822

Figure 5.
Figure 5. Analysis of water in the high resolution structures. A: RMSD plot. B: B-factor plot.

Figure 6.
Figure 6. Ribbon diagram of RNase S coloured according to value of B factor of the main-chain atoms for (A) 5 M and (B) pH 2* structure. Regions of high, intermediate and low B (Å^2) are colored red, white, and blue respectively (scale inset). The figure was generated using the program INSIGHT-II.

The above figures are reproduced from the cited reference with permission from John Wiley & Sons, Inc.

Secondary reference #2

Title

Crystallographic structures of ribonuclease s variants with nonpolar substitution at position 13: packing and cavities.

Authors

R.Varadarajan, F.M.Richards.

Ref.

Biochemistry, 1992, 31, 12315-12327. [DOI no: 10.1021/bi00164a005]

PubMed id

1463720

Secondary reference #3

Title

Thermodynamic and structural consequences of changing a sulfur atom to a methylene group in the m13nle mutation in ribonuclease-S.

Authors

J.Thomson, G.S.Ratnaparkhi, R.Varadarajan, J.M.Sturtevant, F.M.Richards.

Ref.

Biochemistry, 1994, 33, 8587-8593. [DOI no: 10.1021/bi00194a025]

PubMed id

8031793