Accurate
Determination of Interfacial Protein Secondary
Structure by Combining Interfacial-Sensitive Amide I and Amide III
Spectral Signals
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Abstract
Accurate determination of protein
structures at the interface is
essential to understand the nature of interfacial protein interactions,
but it can only be done with a few, very limited experimental methods.
Here, we demonstrate for the first time that sum frequency generation
vibrational spectroscopy can unambiguously differentiate the interfacial
protein secondary structures by combining surface-sensitive amide
I and amide III spectral signals. This combination offers a powerful
tool to directly distinguish random-coil (disordered) and α-helical
structures in proteins. From a systematic study on the interactions
between several antimicrobial peptides (including LKα14, mastoparan
X, cecropin P1, melittin, and pardaxin) and lipid bilayers, it is
found that the spectral profiles of the random-coil and α-helical
structures are well separated in the amide III spectra, appearing
below and above 1260 cm<sup>–1</sup>, respectively. For the
peptides with a straight backbone chain, the strength ratio for the
peaks of the random-coil and α-helical structures shows a distinct
linear relationship with the fraction of the disordered structure
deduced from independent NMR experiments reported in the literature.
It is revealed that increasing the fraction of negatively charged
lipids can induce a conformational change of pardaxin from random-coil
to α-helical structures. This experimental protocol can be employed
for determining the interfacial protein secondary structures and dynamics
in situ and in real time without extraneous labels