Accommodation of amino acid insertions in an alpha-helix of T4 lysozyme. Structural and thermodynamic analysis.


One to four alanines were inserted by site-directed mutagenesis at three different locations within the alpha-helix comprising residues 39 to 50 in bacteriophage T4 lysozyme. All insertion mutants were correctly folded and catalytically active although the insertions led to a thermal destabilization by 1.1 to 4.2 kcal/mol when compared to wild-type. Variants that restored part of the loss in stability associated with the initial alanine insertions could be found by randomizing the inserted amino acids. In selected cases, directed mutagenesis of adjacent residues was also used to regain stability. Structural information obtained from X-ray crystallography and/or 2D-NMR for 10 different variants showed two distinct ways in which the protein responded to the amino acid insertions: (1) The inserted amino acids were incorporated into the helix by replacing preceding wild-type amino acids and causing a shift in register towards the N terminus. As a consequence, wild-type amino acids were translocated from the helix into the preceding loop. (2) Insertions caused a "looping out" within the alpha-helix. In this case the perturbation was confined to a minimal region in the immediate vicinity of the insertion. No change in the length of the helix was detected in either case. The structural response appears to be determined by the maintenance of the hydrophobic interface between the helix and the rest of the protein. This interface remains essentially intact in all variant structures. The results exemplify the plasticity and the adaptability of the protein structure which allows the incorporation of additional amino acids into a secondary structure element without large structural perturbations, as long as vital internal interactions are preserved. They also suggest that loops in proteins related by evolution can vary in length not only because of insertions within the loops themselves but also as a consequence of insertions within neighboring secondary structure elements. Study holds ProTherm entries: 2580, 2581, 2582, 2583, 2584, 14039, 14040, 14041, 14042, 14043 Extra Details: ddG and dTm are relative to cysteine-free pseudo wild type lysozyme,,1L63 (C54T, C97A) insertions; protein evolution; lysozyme; protein flexibility

Submission Details


Submitter: Connie Wang

Submission Date: April 24, 2018, 8:19 p.m.

Version: 1

Publication Details
Heinz DW;Baase WA;Zhang XJ;Blaber M;Dahlquist FW;Matthews BW,J. Mol. Biol. (1994) Accommodation of amino acid insertions in an alpha-helix of T4 lysozyme. Structural and thermodynamic analysis. PMID:8114100
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Endolysin P00720 ENLYS_BPT4