title: Site-directed mutagenesis of the hinge peptide from the hemagglutinin protein: enhancement of the pH-responsive conformational change.
abstract: 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.
authors.affiliation: Center for Engineering in Medicine/Surgical Services, Shriners Hospital for Children, and Harvard Medical School, 51 Blossom Street, Boston, MA, USA. mcasali@med.harvard.edu
authors.forename: Monica
authors.initials: M
authors.lastname: Casali
authors.forename: Scott
authors.initials: S
authors.lastname: Banta
authors.forename: Carlo
authors.initials: C
authors.lastname: Zambonelli
authors.forename: Zaki
authors.initials: Z
authors.lastname: Megeed
authors.forename: Martin L
authors.initials: ML
authors.lastname: Yarmush
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002942:Circular Dichroism; D017931:DNA Primers; D006388:Hemagglutinins / Q000737:chemistry* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation
chemical_list: D017931:DNA Primers; D006388:Hemagglutinins