Sequence of Events during Peptide Unbinding from RNase S: A Complete Experimental Description

Using azobenzene photocontrol to set proteins in motion

“…1d), either isolated [102][103][104][105][106][107] or as secondary structure motifs in larger proteins. 43,58,[64][65][66][67][68][69][70][71][72]75,76 The lightinduced isomerization of an azobenzene molecule around the N=N bond changes the distance between the two anchoring points, and thus controls the α-helical content mechanically. Woolley and coworkers have shown that the helical content is stabilized in the cis-state of the azobenzene relative to that in the trans-state, when the spacing between the two anchoring points of the photoswitch is <9, while it is destabilized for larger spacings (Fig.…”

“…There 77 (c) PDZ3 domain with a photoswitchable α3-helix 64,65 and (d) the RNase S complex with a photoswitchable S-peptide. 43,75 are two approaches to artificially photocontrol a protein and subsequently investigate it with time-resolved methods. First, one can decide to photocontrol a binding partner for the system in question (Fig.…”

“…2b,d). 43,75,77 These partners usually are relatively short peptide ligands, which can be synthesized using standard solid state peptide synthesis and subsequently cross-linked with an azobenzene molecule. This approach is significantly less demanding than the second one, where a photoswitch molecule is inserted postranslationally to a full length protein(Fig.…”

“…30 Technologies to measure transient IR spectra include stepscan FTIR spectroscopy, [31][32][33][34] quantum cascade lasers, [35][36][37][38][39] as well as femtosecond laser based techniques. [40][41][42][43] Combining techniques, one can cover all relevant timescales from 100 fs to seconds and beyond. chemical processes may serve as an inspiration for the more direct design of artificially photocontrollable proteins by involving photoswitchable molecules (or photoswitches for short).…”

“…[51][52][53] By properly incorporating them into a protein system, one may modulate its function or activity directly, [54][55][56][57][58][59][60][61][62][63][64][65] or one may manipulate the binding affinity of a photoswitchable ligand that binds to a protein. 43,[66][67][68][69][70][71][72][73][74][75][76][77][78] Unlike naturally abundant photo-responsive proteins, artificial photocontrol allows for a rational design, depending on the effect one wants to achieve, which can be applied to virtually any protein system. Photoswitches are versatile and fully customizable, and the induced effects are often larger than those achieved with e.g.…”