Investigation of M2 Proton Channel in Membrane by Rapid Laser pH-Jump Technique with TRP Fluorescence as a Probe

Abstract

Protonsplay a central role in many important biological processes such as ATP synthesis, viral infection, and cellular transportation. The mechanism and kinetics of these fast proton-related processes could be studied by the rapid laser-induced pH-jump technique, utilizing photo-acids which can release protons in 20 ns. Many researchers have used the laser pH-jump technique to investigate kinetics of protein folding, acid denature, or EGFP chromophore protonation in sub-millisecond time regime, but there has been no laser pH-jump study about membrane proteins to date, although many proton-related processes such as cellular transportation and ATP synthesis are associated with membrane and channel proteins. In this study, therefore, the laser pH-jump experiment on a membrane protein, Influenza A M2 proton channel (M2), was first demonstrated. In the pH-jump experiment with a membrane, it was observed that the local proton concentration on the membrane surface was higher than bulk solution because of the interaction between membrane and photo-acid, resulting in faster protonation kinetics than in bulk solution. Moreover, we first utilized Trp fluorescence as a probe for laser pH-jump, expanding on its capability. By observing the Trp fluorescence change of M2, sub-millisecond time scale kinetics of M2 activation process was measured upon rapid acidification. Trp fluorescence change could be described by a double exponential decay function, indicating that the first exponential is attributed to histidine protonation and the second is associated with subsequent conformational change. The V27A mutant of M2 showed faster overall kinetics than WT due to the lack of valine neck. We believe that our results advance our mechanistic knowledge of the M2 activation and could pave the way for the investigation of dynamics, kinetics, and mechanism of other proton-related processes.