Factors contributing to decreased protein stability when aspartic acid residues are in {beta}-sheet regions.

Journal Article · Mon Jul 01 04:00:00 EDT 2002 · Protein Sci.

DOI:https://doi.org/10.1110/ps.4920102· OSTI ID:949497

Pokkuluri, P R; Cai, X; Raffen, R; Gu, M; Stevens, F J; Schiffer, M

Asp residues are significantly under represented in {beta}-sheet regions of proteins, especially in the middle of {beta}-strands, as found by a number of studies using statistical, modeling, or experimental methods. To further understand the reasons for this under representation of Asp, we prepared and analyzed mutants of a {beta}-domain. Two Gln residues of the immunoglobulin light-chain variable domain (V{sub L}) of protein Len were replaced with Asp, and then the effects of these changes on protein stability and protein structure were studied. The replacement of Q38D, located at the end of a {beta}-strand, and that of Q89D, located in the middle of a {beta}-strand, reduced the stability of the parent immunoglobulin VL domain by 2.0 kcal/mol and 5.3 kcal/mol, respectively. Because the Q89D mutant of the wild-type V{sub L}-Len domain was too unstable to be expressed as a soluble protein, we prepared the Q89D mutant in a triple mutant background, V{sub L}-Len M4L/Y27dD/T94H, which was 4.2 kcal/mol more stable than the wild-type V{sub L}-Len domain. The structures of mutants V{sub L}-Len Q38D and V{sub L}-Len Q89D/M4L/Y27dD/T94H were determined by X-ray diffraction at 1.6 A resolution. We found no major perturbances in the structures of these QD mutant proteins relative to structures of the parent proteins. The observed stability changes have to be accounted for by cumulative effects of the following several factors: (1) by changes in main-chain dihedral angles and in side-chain rotomers, (2) by close contacts between some atoms, and, most significantly, (3) by the unfavorable electrostatic interactions between the Asp side chain and the carbonyls of the main chain. We show that the Asn side chain, which is of similar size but neutral, is less destabilizing. The detrimental effect of Asp within a {beta}-sheet of an immunoglobulin-type domain can have very serious consequences. A somatic mutation of a {beta}-strand residue to Asp could prevent the expression of the domain both in vitro and in vivo, or it could contribute to the pathogenic potential of the protein in vivo.

• Research Organization: Argonne National Laboratory (ANL)
• Sponsoring Organization: SC; OGA
• DOE Contract Number: AC02-06CH11357
• OSTI ID: 949497
• Report Number(s): ANL/BIO/JA-41500
• Journal Information: Protein Sci., Journal Name: Protein Sci. Journal Issue: 7 ; Jul. 2002 Vol. 11
• Country of Publication: United States
• Language: ENGLISH

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
99 GENERAL AND MISCELLANEOUS
ASPARTIC ACID
IN VITRO
IN VIVO
PROTEIN STRUCTURE
PROTEINS
RESIDUES
SOMATIC MUTATIONS
STABILITY
X-RAY DIFFRACTION