(1) Centre
for Biotechnology, University of Turku, P.O. Box 123, Turku, Finland
(2) Biochemistry and Microbiology, Cook College, Rutgers University, P.
O: Box231, New Brunswick, NJ 08903, USA
Having noticed that 65% of the charged surface area of muconate lactonising enzyme (MLE) accessible in the extended form of the protein is buried in the folded monomer (1), we decided to re-examine the burial of charged surface in proteins. To our surprise, MLE is not unusual: many proteins of sizes 400-700 residues bury equivalently large proportions of their charged surface. Like aliphatic, aromatic and polar uncharged (chiefly peptide backbone) surface (2), the percentage burial of polar charged surface also increases with protein size. The burial of each surface with size obeys an equation of the form (100% - aLb) where L is the sequence length. Furthermore, the rate of percentage burial for charged surface is significantly larger than that for polar uncharged and aromatic surface, and slightly larger than that for aliphatic surface, especially in the size range 100-300 residues. Large proteins thus have a significantly larger amount of destabilisation by buried charge than smaller ones.
We therefore examined some representative proteins to see how the charged surface was buried: were all residues somewhat buried?, were the buried residues primarily in the active site as part of "electrostatic strain"? In general, as previously observed (2), all proteins completely bury (<5% accessible) a small percentage of their charged residues; however proteins often have many substantially buried (5-30% accessible) charged residues. These residues are scattered throughout the molecule. If, as recent theoretical and mutagenesis studies have suggested (3, 4) buried charged interactions are not stabilising, our results imply that "electrostatic strain" may contribute substantially to the destabilisation of the native conformation.
The burial of charged surface has two aspects: as it scales smoothly with size, it is a non-specific contributor to the DG of unfolding, like conformational entropy. However, unlike conformational entropy, it is also specific, in that specific residues are buried to specific extents in specific places. Consequently, burial of charged residues may be an important way by which proteins adjust the balance between flexibility and stability, or by which they stabilise the native conformation over other related conformations (3). If so, modifying such residues outside the active site offers another way to engineer (thermo)stability into proteins.
(1) Helin, S., Kahn, P.C., Guha, B.L., Mallows, D.L. & Goldman, A. J. Molec. Biol., 254, 918-941 (1995).
(2) Miller, S., Janin, J., Lesk, A. M. & Chothia, C. J. Molec. Biol., 196, 641-656 (1987).
(3) Hendsch, Z.S. & Tidor, B. Prot. Sci., 3, 211-226 (1994).
(4) Waldburger, C.D., Schildbach, J.F. & Sauer, R.T. Nature Struct. Biol., 2, 122-128 (1995).