Toward a simplification of the protein folding problem: a stabilizing polyalanine alpha-helix engineered in T4 lysozyme.
In an attempt to simplify the protein folding problem, and also to further investigate the role of alanine as a helix-stabilizing residue, a series of alanines was introduced within the alpha-helix that includes residues 126-134 of T4 lysozyme. In wild-type lysozyme this alpha-helix contains alanine residues at positions 129, 130, and 134. Mutant lysozymes with alanines substituted at positions 128, 131, 132, and 133, either as single substitutions or in selected combinations, were constructed by oligonucleotide-directed mutagenesis. With the exception of the replacement of Leu 133, which is buried within the hydrophobic core of the protein, all the variants were more stable than wild-type lysozyme. The variant with alanines substituted at positions 128, 131, and 132 (E128A/V131A/N132A), which incorporates the sequence Ala 128-Ala 129-Ala 130-Ala 131-Ala 132-Leu 133-Ala 134, has a melting temperature 3.3 degrees C above that of wild-type lysozyme. Determination of the crystal structure of this mutant lysozyme shows that the replacement of Glu 128, Val 131, and Asn 132 with alanine causes alpha-helix 126-134 to rotate 3.4 degrees about an axis parallel to its own axis. This rotation seems to be triggered primarily by the loss of a hydrogen bond between Asn 132 and Ser 117 and is associated with the repacking of several side chains at the interface between alpha-helix 126-134 and the adjacent alpha-helix 115-122.(ABSTRACT TRUNCATED AT 250 WORDS)
Zhang
X J
XJ
Institute of Molecular Biology, University of Oregon, Eugene 97403.
Baase
W A
WA
Matthews
B W
BW
D000409:Alanine; D000595:Amino Acid Sequence; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics*; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013604:T-Phages / Q000201:enzymology*; D013816:Thermodynamics; D014961:X-Ray Diffraction
D009113:Muramidase; D000409:Alanine