PEDS Advance Access published online on April 14, 2008
Protein Engineering Design and Selection, doi:10.1093/protein/gzn018
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Site-directed mutagenesis of the hinge peptide from the hemagglutinin protein: enhancement of the pH-responsive conformational change
1Center for Engineering in Medicine/Surgical Services, Shriners Hospital for Children, Massachusetts General Hospital, and Harvard Medical School, 51 Blossom Street, Boston 2Molecular and Cellular Biology Department, Harvard University, Cambridge, MA, USA
5 To whom correspondence should be addressed. E-mail: mcasali{at}med.harvard.edu; ireis{at}sbi.org
Environmentally responsive proteins and peptides are increasingly finding utility in various engineered systems due to their ability to respond to the presentation of external stimuli. A classic example of this behavior is the influenza hemagglutinin (HA) fusion protein. At neutral pH, HA exists in a non-fusogenic state, but upon exposure to low pH, the conformation of the structure changes to expose a fusogenic peptide. During this structural change, massive rearrangements occur in a subunit of HA (HA2). Crystallography data has shown that a loop of 28 amino acids (residues 54–81) undergoes a dramatic transition from a random coil to an alpha-helix. This segment connects to two flanking helical regions (short and long) to form a long, continuous helix. Here, we report the results of site-directed mutagenesis study on LOOP-36 to further understand the mechanism of this important stimulus-responsive peptide. The conformational transition of a bacterially expressed LOOP-36 was found to be less dramatic than has been previously reported. The systematic mutation of glutamate and histidine residues in the peptide to glutamines (glutamine scanning) did not impact the conformational behavior of the peptide, but the substitution of the glycine residue at position 22 with alanine resulted in significant pH-responsive behavior. Therefore this mutant stimulus-responsive peptide may be more valuable for future protein engineering and bionanotechnology efforts.
Keywords: circular dichroism/conformational change/hemagglutinin/site-directed mutagenesis/stimulus-responsive
Received January 4, 2008; revised March 6, 2008; accepted March 16, 2008.
3 Present address: Department of Chemical Engineering, Columbia University, New York, NY, USA
4 Present address: Department of Biomedical Engineering, Rutgers University, Piscataway, NJ, USA