Protein structural plasticity exemplified by insertion and deletion mutants in T4 lysozyme

Abstract

To further investigate the ways in which proteins respond to changes in the length of the polypeptide chain, a series of 32 insertions and five deletions were made within nine different a-helices of T4 lysozyme. In most cases, the inserted amino acid was a single alanine, although in some instances up to four residues, not necessarily alanine, were used. Different insertions destabilized the protein by different amounts, ranging from approximately 1 to 6 kcal/mol. In one case, no protein could be obtained. An "extension" mutant in which the carboxy terminus of the molecule was extended by four alanines increased stability by 0.3 kcal/mol. For the deletions, the loss in stability ranged from approximately 3 to 5 kcal/mol. The structures of six insertion mutants, as well as one deletion mutant and the extension mutant, were determined, three in crystal forms nonisomorphous with wild type. In all cases, including previously described insertion mutants within a single a-helix, there appears to be a strong tendency to preserve the helix by translocating residues so that the effects of the insertion are propagated into a bend or loop at one end or the other of the helix. In three mutants, even the hydrophobic core was disrupted so as to permit the preservation of the a-helix containing the insertion.Translocation (or "register shift") was also observed for the deletion mutant, in this case a loop at the end of the helix being shortened. In general, when translocation occurs, the reduction in stability is only moderate, averaging 2.5 kcal/mol. Only in the most extreme cases does "bulging" or "looping-out'' occur within the body of an a-helix, in which case the destabilization is substantial, averaging 4.9 kcal/mol. Looping-out can occur for insertions close to the end of a helix, in which case the destabilization is less severe, averaging 2.6 kcallmol. Mutant A73-[AAA], as well as mutants R119-[A] and V131-[A], include shifts in the backbone of 3-6 A, extending over 20 residues or more. As a result, residues 114-142, which form a "cap" on the carboxy-terminal domain, undergo substantial reorganizations such that the interface between this "cap" and the rest of the protein is altered substantially. In the case of mutant A73-[AAA], two nearby a-helices, which form a bend of approximately 105 degrees in the wild-type structure, reorganize in the mutant structure to form a single, essentially straight helix. These structural responses to mutation demonstrate the plasticity of protein structures and illustrate ways in which their three-dimensional structures might change during evolution.

Engineering Soluble Monomeric Streptavidin with Reversible Biotin Binding Capability

“…To engineer monomeric streptavidin with a minimal number of mutated residues, an attractive approach is to introduce both charge repulsion and steric hindrance at these interfaces. As protein has structural plasticity (23)(24)(25), it is vital to select interfacial residues located on a rigid surface to maximize the effects of charge repulsion and steric hindrance. Because streptavidin subunit forms an eightantiparallel stranded ␤-barrel structure (4, 5), the selected …”

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

“…If insertions (6,7,29,30) of a small number of amino acids (e.g., 1-3) are made within ␣-helices, the polypeptide chain often is translocated, thereby preserving the overall helical region (6,7,31). Sometimes the length of the ␣-helix remains the same (6,7).…”

“…Sometimes the length of the ␣-helix remains the same (6,7). In other cases it can be lengthened, corresponding to the added amino acids (31). These observations suggest that the structures of helical segments within proteins are determined by a compromise between the sequence of amino acids within the helix and the structural context provided by the rest of the protein.…”

Investigating protein structural plasticity by surveying the consequence of an amino acid deletion from TEM‐1 β‐lactamase

“…Therefore, long flexible loops are considered the places most likely to tolerate a deletion [3]. However, analysis of several specific deletion mutants of T4 lysozyme [8], the B1 domain of protein G [9] and Ricin A-chain [10] suggests this is not always the case. The difficulty in predicting the consequence of a deletion mutation means that most protein engineering and design efforts aimed at investigating or altering a protein's properties tend to ignore them [11].…”