Conformational changes of a membrane protein determined by infrared difference spectroscopy beyond the diffraction limit

Optogenetics is a revolutionary method for studying neural activity by exploiting the optical stimulation of neurons made possible by artificial genetic expression of light-sensitive transmembrane "gate" proteins. Evidence of functional conformational changes of the gate proteins can be obtained by difference infrared (IR) absorption spectroscopy triggered by light stimuli. Here we investigate the effect on the photocycle of the prototype optogenetic protein channelrhodopsin (ChR2) of gold surfaces placed in close proximity to the protein molecules. In order to do this, we bring difference IR spectroscopy to the nanoscale, using a platform based on the coupling of a tunable mid-IR quantum cascade laser and an atomic force microscope (AFM). Sensitivity to individual subwavelength-sized membrane patches, embedding fewer than a hundred ChR2 molecules, is achieved by taking advantage of a plasmonic field enhancement in the nanogap between the gold-coated AFM tip and an ultraflat gold surface used as a sample support. We obtain relative difference absorption variations smaller than 10(-2) and benchmark nanospectroscopy difference spectra against those taken with the Fourier transform IR (FTIR) spectroscopic technique on the same sample. We identify distinct simultaneous processes in the ChR2 photocycle by performing singular value decomposition of the FTIR difference spectra, and use this procedure to compare IR nanospectroscopy data on individual membrane patches in contact with gold surfaces with those obtained on thick stacks of membrane patches unexposed to metal surfaces. ChR2 proteins maintain their gate function when placed in a 14-nm-wide gap between two gold surfaces, apart from minor modifications to their kinetics. Our results are relevant to optogenetic applications that require physical contact between nanoscale metallic probes and electrodes and the membrane of single neuronal cells. More generally, our work paves the way towards the spectroscopic study of transmembrane proteins at the nanoscale.