Structural Consequences of an Amino Acid Deletion in the B1 Domain of Protein G Karyn T. O’Neil, 1,2 Alvin C. Bach II, 2 and William F. DeGrado 1 * 1 Department of Biochemistry and Biophysics, The University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 2 Departments of Applied Biotechnology and Chemical and Physical Science, DuPont Merck Pharmaceutical Company, Wilmington, Delaware ABSTRACT We describe the NMR structure of a deletion mutant of the B1 IgG-binding domain from Group G Streptococcus. The deletion occurs within the last -strand of the protein, where it may potentially have a deleterious effect on the stability of the protein if the protein were not able to confor- mationally adjust to the perturbation. In particular, the deletion changes the registry of the final three residues in the sheet, forcing a polar Thr to be buried in the interior of the protein and exposing a hydrophobic Val to solvent. The deletion could also potentially create a large cavity in the -sheet and force the - and -carboxylates of the C-terminal Glu residue into a partially buried region of the sheet. The structure of the mutant illustrates how the conformation of the protein adjusts to the deletion, thereby mitigating some of the potentially deleteri- ous consequences. Although the elements of second- ary structure are retained between the mutant and the wt domain, there are multiple small adjustments in the segments connecting secondary structure elements. In particular, a hydrogen bond between the Glu57 carboxylates and two main chain amides is introduced that alters the conformation in the loop connecting the helix to strand 3. In addition, to minimize hydrophobic surface exposure, the turn connecting strands 1 and 2 folds toward the core so that the molecular volume is decreased. Proteins 2000;41:323–333. © 2000 Wiley-Liss, Inc. Key words: protein stability; deletion mutant; pro- tein G INTRODUCTION Mutational analysis provides the protein engineer with a powerful tool for answering questions about protein folding. By introducing site-directed changes at specific positions in a protein sequence it is possible to test hypotheses about protein stability and function. Many biochemical and genetic studies have measured the effects of amino acid mutations in a wide variety of proteins. Such studies provide a basis for our understanding of secondary structure stabilization, 1–5 electrostatic interactions, 6,7 and hydrophobic packing interactions in proteins. 8,9 Mutations that involve insertion or deletion of amino acids are less well studied. However, an analysis of families of homologous proteins suggests that evolution of closely related proteins is often the result of insertion or deletion mutations. 10 Therefore, an understanding of the effects of such mutations may provide novel possibilities for altering protein structure and function by design. 11,12 Experimentally, the expectation that insertion or deletion of amino acid residues will severely compromise the stabil- ity of the protein is not always borne out. For example, it is possible to introduce additional alanine or glycine residues at many different positions including helices, turns, and strands of staphylococcal nuclease 13 or T4 lysozyme. 14 Often, inclusion of the additional amino acids has little effect on the stability of the molecule because residue insertions are often readily accommodated by generation of local bulges or lateral displacements that shift the packing register. 14 –16 Fewer systematic studies of the effects of amino acid deletions have been reported. It may be anticipated that this class of mutations will generate proteins with signifi- cantly altered stability and properties. However, the struc- tures of such mutants indicate that they are often well packed and have normal hydrogen bonding patterns. The crystal structure of a six amino acid deletion mutant of staphylococcal nuclease illustrates the serious folding implications for deletion of residues linking secondary structure elements. 17 This protein mutant exhibits dimeric behavior, although the parent protein is monomeric. Alter- natively, an engineered mutant of triose phosphate isomer- ase has been shown to exhibit monomeric instead of the native dimeric behavior. 18 One of the most well-studied cases of a dysfunctional deletion mutation is in the cystic fibrosis transmembrane conductance regulator. Deletion of phenylalanine 508 and the concomitant loss of function accounts for approximately 70% of the disease alleles. 19 Deletion of Phe508 induces a decrease in -structure, an increase in random coil structure, 19 and decreased stabil- ity. The B1 IgG-binding domain from Group G Streptococcus has been used extensively as a model system for answering many questions about protein folding. The structure of the domain is well characterized by both NMR 20,21 and crystal- lographic methods. 22 It is a small compact domain that Alvin C. Bach II’s present address is Discovery Analytical Chemis- try, Wyeth Ayerst Pharmaceutical Company, CN8000, Princeton, NJ 05843-8000. *Correspondence to: William F. DeGrado, Department of Biochemis- try and Biophysics, Philadelphia, PA 19104-6059. E-mail: wdegrado@mail.med.upenn.edu Received 12 August 1998; Accepted 11 May 2000 PROTEINS: Structure, Function, and Genetics 41:323–333 (2000) © 2000 WILEY-LISS, INC.