Research Illustration #1 - Photoswitchable Fluorescent Proteins

Photoactivation, photoconversion and photoswitching are all categorized as optical highlighting, and these exciting properties further expand the FP (fluorescent protein) toolkit for bioimaging and molecular biology. They can help tremendously about photolabeling and tracking fusion proteins in a living cell, e.g. using photoswitchable FPs to tag a protein and turning it off to avoid photobleaching, and turning it on at a specific time point to find out the precise location of the tagged protein inside the cell. The protein trafficking will then be mapped. This easily finds its usage in super-resolution fluorescence microscopy, single-molecule photonic devices, optical nanoscopy, biotechnology, drug discovery and delivery, and far beyond.

FSRS spectroscopy is well suited to study those photoswitchable FPs after the successful proof-of-concept experiment on the wtGFP fluorescence mechanism (see Publications 2.). The simultaneously high spectral (spatial) and temporal resolution has enabled us, for the first time, to demonstrate a femtosecond phenolic-ring wagging motion of the chromophore in gating the picosecond excited-state proton transfer (ESPT) inside the protein pocket. This level of structural dynamics insight in the very early time regime immediately following photoexcitation offers us a perfect paradigm in visualizing quantum coherence in play, in deciphering the multidimensional chemical reaction coordinate in living biological systems due to their specific potential energy landscape, and in understanding conformational dynamics in determining the reaction outcome/biological function on their native timescales. Nothing comes out of the blue so the answers to these questions that are quite fundamental in nature and highly relevant for practical applications will be sweet. The advantage of my group in contributing to the discussion here is to stand in the unique interface of chemistry, physics and biology to unearth something new about the biomolecules, something deep about the process, and to develop a comprehensive understanding and grasp of the fascinating biofunctions ── no matter how diverse they are.

These novel FPs are responsive to external light excitations and subsequently transitioning between different conformational states. The chromophore that is responsible for the overall protein color is normally protected from the bulk solvent and strategically located in the central region of the protein matrix (see figure below). The chromophore absorbs the incoming photons and distributes the excitation energy around. Certain conformational states are preferred over the others due to steric constraints, potential energy landscape, and electrostatic interactions particularly hydrogen bonding with nearby protein residues. Therefore the essential questions for us to answer are: (1) What is the typical timescale for excited-state structural evolution of the FPs, and is there a ubiquitous molecular motion involved? (2) How does the protein matrix react to the chromophore conformational dynamics and how does the protein matrix stabilize a specific conformation? (3) What are the "weakest" links or spots for excited-state structural evolution to begin with, and later to stop? (4) Which residues and protein-chromophore interactions are key to engineer for higher quantum efficiency in reaching a desirable state with great photostability?

Ultrafast vibrational spectroscopy is poised to elucidate the photochemical reaction mechanism of a novel group of photoswitchable FPs. (a) Reversible photoswitching of the asFP595-A143S protein crystal observed under a fluorescence microscope. The resting state is barely fluorescent. (b) Brightfield image of the asFP595-A143S crystal where an additional H-bond between the MYG chromophore phenol ring and S143 stabilizes the fluorescent cis conformer. (c) On/off cycles of the asFP595-A143S crystal fluorescence by switching between green and much weaker blue light. Adapted from Andresen, M., et al. PNAS 102 (2005) 13070. (d) The sea anemone Anemonia sulcata that the deep purple-colored asFP595 is cloned from. (e) The strategic position of the three-residue chromophore in the central region of the protein matrix where the chromophore-protein interaction is key in determining the structural dynamics of the overall FP. A zoom-in of the immediate vicinity of the chromophore is shown below the top-view-down of the protein barrel structure, in which the proposed molecular photoswitching in this KFP (kindling FP) involves structural transition between the fluorescent "on" state (cis form, A) and "off" state (trans form, B), on the ps timescale. Adapted from the X-ray crystallographic study, Henderson, J. N. and Remington, S. J. Physiology 21 (2006) 162. A potential issue with asFP595 is the Q.Y. of the activated state is very low (<0.12) whereas the Q.Y. for GFP is ~0.8. (f) Another sea coral reef that contains the naturally tetrameric Clavularia CFP, from which the monomeric teal FP0.7 (mTFP0.7) is derived. (g) The architecture and dimension of the GFP-like FP viewing from the top of the protein. The embedded chromophore is shielded from the bulk solvent and the diameter of the peripheral beta-barrel is ~2 nm. So this is nanobiotechnology! (h) The excited-state proton transfer (ESPT) chain involving the wtGFP chromophore in action has been recently exposed by the FSRS technique, in which the fs phenolic ring wagging motion gates the ps ESPT. Adapted from Fang, C., et al. Nature 462 (2009) 200. The vivid details of the structural evolution of the chromophore in association with its protein surroundings immediately following photoexcitation reveal the intrinsic multidimensional reaction coordinate in GFP, likely ubiquitous among other FPs and photosensitive materials. (i) A molecular depiction of the chromophore atomistic motions typically observed in FSPS spectra as these high-frequency bond stretching modes lead to higher Raman polarizability and generally stronger spectral features. The phase relationship between the time-dependent peak variations is directly reporting on the multimode interaction across the chromophore rings, hence the anharmonicities and the potential energy landscape of the overall molecule. (j) Photoswitching of the mTFP0.7 chromophore inside the protein pocket. Upon 458 nm excitation, the fluorescent "on" state (shown on the right, a cis conformer) quickly converts to the dark "off" state (left) where the chromophore is protonated and in a trans configuration. The chromophore is significantly nonplanar and the cyan dashed line represents the adjacent protein residues E148, I161, Y181 and an internal water molecule. Upon 400 nm excitation, fluorescence is quickly restored in a pH-independent manner when the phenolate ring loses its proton and shifts to a different location inside the protein pocket, facilitated by a new set of boarder residues S146, H163 and another internal water molecule (represented by the cyan dashed line). There is indeed significant alteration of the electrostatic interaction and H-bonding pattern in the immediate vicinity of the chromophore during photoswitching, and once we gain more mechanistic insights on these photoinduced structural transitions from FSRS, we can further refine and develop the FP toolkit for molecular and cellular biology, with higher Q.Y., better photostability, and more versatile control of the photochromic properties of FPs.