Hemagglutinin TMD peptide inserts into membrane bilayer at acidic and neutral pH. (A) The blue shift and enhancement of fluorescence intensity of tryptophan residues in TMD when incubated in DMPC:DMPG vesicles attest to the location of TMD in the membrane hydrophobic milieu. The emission maximum for tryptophan in an aqueous environment is 350 nm. (B) K
SV acrylamide quenching measurements also indicate deep insertion of TMD into the membrane interior. The dramatic decrease in K
SV in the vesicular dispersion compared with that in PB buffer shows that tryptophan side chains are embedded deep into the membrane. Moreover, a twofold reduction in K
SV, as well as decreased K
SV on neutralization, upon incubating in PC:PG vesicles at pH 5.0 compared with that at pH 7.4 suggests that the TMD penetration is deeper at acidic pH.