Optical switching of dipolar interactions on proteins

Abstract

This work shows that optical switching between the spiro (SP) and merocyanine (MC) states of different photochromes specifically labeled to G-actin can be used to rapidly and reversibly modulate specific dipolar interactions within the conjugate. Members of a common spirobenzopyran photochrome and a related spironaphthoxazine that differ only in the locations of their alkylating groups were selectively labeled to Cys-374 on G-actin. The nature of MC and SP interactions within G-actin was investigated by using optical spectroscopy. The average absorption energy of the highly polarized MC is sensitive to interactions with polar groups on solvents and G-actin; the average absorption energy of the corresponding SP state was found to be relatively constant, consistent with its lower dipole moment compared with MC (5 and 20 D, respectively). Alternate excitation of spirobenzopyran G-actin conjugates with 365 and 546 nm leads to rapid transitions from the SP to MC states and MC to SP states, respectively; optical switching within spirobenzopyran-G-actin occurs with high fidelity and the recovery of specific dipolar interactions between the protein and the MC and SP states. The difference in the free energy for specific dipolar interactions between different MC states within G-actin (6 kcal͞mol) is similar to that found for complexes of G-actin and its regulatory proteins. We propose, therefore, that optical switching between SP and MC within an appropriately labeled conjugate could be used to inhibit a functional interaction with a ligand in the MC, but not the SP, state. protein engineeringT he increasing need to understand biomolecular and cellular processes in terms of the mechanisms of protein-mediated reactions is driving the development of new optical probes capable of specifically manipulating and mapping protein interactions and activities in complex environments (1-6). We have previously shown that protein interactions and activities can be manipulated in vitro and in vivo by using light-directed activation of caged proteins (3,6). This perturbation technique can yield important kinetic and functional information on the protein with high temporal and spatial resolution (6). However, the photoisomerization of the 2-nitrophenyl group limits the usefulness of this technique because the reaction is irreversible, slow, and generates toxic photoproducts.We propose a previously undescribed approach for optical switching of protein interactions (7) that is based on optical control of specific dipolar interactions within proteins specifically labeled with a single photochromic probe. The spirobenzopyran probe is well suited for this task because it undergoes rapid, and reversible, light-directed transitions between a colorless spiro (SP) state and a colorful merocyanine (MC) state without the release of photoproducts (7-13). Optical spectroscopy is used to characterize the interactions of the two switch states within the conjugates and to provide information on their dipolar interactions with G-actin. We argue that if ...

Optical lock-in detection imaging microscopy for contrast-enhanced imaging in living cells

“…The reproducibility of the increase and decrease in fluorescence demonstrates that nitro-BIPS undergoes high-fidelity optical switching and is largely resistant to fatigue. From previous studies (17,18), we know that the time constant for thermally driven (dark) transition of MC to SP is Ϸ3,000 s for protein-bound nitroBIPS, i.e., slow enough to enable nitroBIPS to function as an all-optically controlled switch. Thus, nitroBIPS-derived probes undergo 1-and 2-photon-driven, rapid, efficient, and high-fidelity optical switching within living cells using excitation intensities that are commonly used without toxicity in live-cell microscopy.…”

“…Employing this concept, we developed optical lock-in detection (OLID) as a means for contrast-enhanced imaging of probes within living cells. We demonstrate OLID on a chemical probe, nitrospirobenzopyran (nitroBIPS; [17][18], and on a fluorescent protein, Dronpa (19), both of which can be driven optically in a reversible manner between nonfluorescent and fluorescent states. These correspond respectively to the spiro (SP) and merocyanine (MC) states for nitroBIPS and the trans and cis states for Dronpa.…”

“…OLID using optical switch probes described herein affords key advantages for high-contrast imaging: (i) the mechanisms of optical switching of nitroBIPS (17)(18)(20)(21) and related photochromes (22) and Dronpa (19,23) are known, and do not require chemical additives; (ii) optical switching between the two states (Ͻ2 s) is much faster than probes used in PALM and STORM; (iii) optically driven transitions occur in the entire population of probe molecules, providing large signals and rapid imaging; and (iv) kinetic signatures and quantum yields for excited-state transitions can easily be measured in the sample and are used for lock-in detection. Thus, the properties of optically switched organic dyes and fluorescent proteins in OLID are compatible with rapid imaging of specific proteins and structures in live cells and within live animals and can be readily used in most laboratories on existing microscopes.…”

“…We first examined the ability of light to modulate the fluorescence of two synthetic switches within live cells, the thiol-reactive probe 8-iodomethyl-nitroBIPS (17)(18) and the membrane probe C11-nitroBIPS (Fig. 1A).…”

Reversible optical control of cyanine fluorescence in fixed and living cells: optical lock-in detection immunofluorescence imaging microscopy

“…(b) Synthesis of optical switch probes Details will be presented elsewhere, although the synthetic methods used for the preparation of Cy-NISO, Cy-NISO-Nhydroxysuccinimide ester (NHS), Cy3-NISO-benzylguanosine (BG) and NISO-NHS are similar to those used in our earlier papers [18,19].…”