Conformational States and Transitions in Green Fluorescent Protein Chromophore Studied by Single Molecule Sers

Abstract

Green fluorescent protein (GFP) from Aequorea victoria won the Nobel Prize in 2008 for its revolutionary impact on bio-imaging. However, conformational states of its chromophore as well as transitions between them are not understood well. Using "nanometal-on-semiconductor" SERS substrates, the present work acquires the vibrational spectra of single GFP molecules under 532 nm laser excitation. Once an aliquot of 10-9 M GFP is spotted on a SERS substrate and a Raman acquisition is started, intense and well-resolved peaks are observed to appear and disappear repeatedly over a weak background. These temporal "spectral jumps" are captured in every half a minute on the average and sustain for 1 s or less. Each jump is associated with a single GFP molecule diffusing into a high SERS enhancement factor site (i.e., a "hotspot"), residing it for 1 s or less, and eventually diffusing out of it. The SERS is acquired in time series at continuous intervals of 50-100 ms. A minimum of 4 conformational states of the GFP chromophore were observed as cis/trans and protonated/deprotonated combinations. Statistical analysis of the GFP populations at different states reveals substantial presence of all 4 states. Among the 4 states, the transitions occur in between certain pairs of states that line-up in a cyclic pattern. Further, as inferred from the calculated transition probabilities, particular transitions become more probable under 532 nm excitation. Consequently, this bias leads to the increased population of certain states that is pronounced more with increasing laser intensity.