Quantitative Analysis of the Effects of Photoswitchable Distance Constraints on the Structure of a Globular Protein

Abstract

Photoswitchable distance constraints in the form of photoisomerizable chemical cross-links offer a general approach to the design of reversibly photocontrolled proteins. To apply these effectively, however, one must have guidelines for the choice of cross-linker structure and cross-linker attachment sites. Here we investigate the effects of varying cross-linker structure on the photocontrol of folding of the Fyn SH3 domain, a well-studied model protein. We develop a theoretical framework based on an explicit-chain model of protein folding, modified to include detailed model linkers, that allows prediction of the effect of a given linker on the free energy of folding of a protein. Using this framework, we were able to quantitatively explain the experimental result that a longer, but somewhat flexible, cross-linker is less destabilizing to the folded state than a shorter more rigid cross-linker. The models also suggest how misfolded states may be generated by cross-linking, providing a rationale for altered dynamics seen in nuclear magnetic resonance analyses of these proteins. The theoretical framework is readily portable to any protein of known folded state structure and thus can be used to guide the design of photoswitchable proteins generally.

A bisazobenzene crosslinker that isomerizes with visible light

“…While cis lifetimes may be longer when the crosslinker is attached to a biomolecule, it is likely that this crosslink will predominantly cycle between the trans , trans and trans , cis -species under low irradiation intensities. These species have overlapping end-to-end distances and would not be expected to drive significant conformational change in a single attached biomolecule [6]. However, high (e.g., pulsed) light intensities may be expected to populate the cis , cis -state and may permit pulsed-light-driven conformational changes.…”

“…A quantitative analysis of the effects of crosslinkers on protein folding led to the conclusion that photocontrol of folding is best achieved by using rigid crosslinkers that undergo a large change in end-to-end distance upon photoisomerization [6]. Previously, we reported the design and synthesis of the rigid bisazobenzene crosslinker BPDBS, (4,4'-bis(4-(2-chloroacetamido)phenyl)diazenylbiphenyl-2,2'-disulfonate) ( 1 , Scheme 1) [24].…”

Direct and Versatile Synthesis of Red‐Shifted Azobenzenes

“…[5] To realize light-responsive systems at the molecular level, photoswitchable compounds are utilized that show ad istinctive change in their properties upon irradiation. [1] Interesting applications of molecular switches for chemical biology were showcased in protein/ peptide control [6][7][8] and photopharmacology, [9,10] allowing the control of drug activity in cancer chemotherapy, [11,12] neurology, [13] and antibiotic treatment, [14] amongst others.T oachieve effective and reversible control upon irradiation, azobenzenebased molecular photoswitches [1-3, 5, 7, 10, 12, 15, 16] have been extensively used because of their large change in geometry and dipole moment upon photo-isomerization from the trans to the cis isomer ( Figure 1A). Tr aditionally,t his trans-cis isomerization is achieved with UV-light irradiation, whereas the reverse cis-trans isomerization can be evoked by either thermal relaxation or visible-light irradiation.…”

Red, far-red, and near infrared photoswitches based on azonium ions

“…26 The bicyclic pyrrolidine version of 2 proved to be insoluble so that it was replaced by the monocyclic amino-pyrrolidine in that case. 26 The bicyclic pyrrolidine version of 2 proved to be insoluble so that it was replaced by the monocyclic amino-pyrrolidine in that case.…”