Photocontrolling Protein–Peptide Interactions: From Minimal Perturbation to Complete Unbinding

Abstract

An azobenzene-derived photoswitch has been covalently cross-linked to two sites of the Speptide in the RNase S complex in a manner that the alpha-helical content of the S-peptide reduces upon cis-to-trans isomerization of the photoswitch. Three complementary experimental techniques have been employed, isothermal titration calorimetry, circular dichroism spectroscopy and intrinsic tyrosine fluorescence quenching, to determine the binding affinity of the S-peptide to the S-protein in the two states of the photoswitch. Five mutants with the photoswitch attached to different sites of the S-peptide have been explored, with the goal to maximize the change in binding affinity upon photoswitching, and to identify the mechanisms that determine the binding affinity. With regard to the first goal, one mutant has been identified, which binds with reasonable affinity in the one state of the photoswitch, while specific binding is completely switched off in the other state. With regard to the second goal, accompanying molecular dynamics simulations combined with a quantitative structure activity relationship revealed that the alpha-helicity of the S-peptide in the binding pocket correlates surprisingly well with measured dissociation constants. Moreover, the simulations show that both configurations of all S-peptides exhibit quite well-defined structures, even in apparently disordered states.

Light-controllable dithienylethene-modified cyclic peptides: photoswitching the in vivo toxicity in zebrafish embryos

“…Analyzing our data, it was interesting to see that the structure-toxicity relationships turned out to be more complicated than it had initially been anticipated and implemented in the original design of the peptide series. Nonetheless, except for the peptides in series iv (16-18, with increased charge densities) and one of the homodimers (19), the difference in activity between the ring-open and the ring-closed photoforms was significant. In all cases, the ring-open states were more active than the corresponding ring-closed photoforms.…”

“…The data in Table 2 reveals several structure-toxicity correlations. Both of the tested dimers (19,20) showed relatively inefficient photoactivation. Given that 19 and 20 were observed to photoisomerize readily between the ring-closed and ringopened isomers, we can presume that homodimerization may affect the way in which the peptides interact with their molecular targets, or it may compromise their long-term biostability.…”

“…In recent years, photopharmacology [14][15][16] has emerged as a successful approach to enhance spatiotemporal selectivity of chemotherapeutics, decreasing the overall toxicity and increasing the safety of drugs. Various compound classes are currently being explored as photopharmacology agents [16][17][18][19][20][21], including peptides [19][20][21]. The idea behind photopharmacology is based on the design and use of drugs containing a reversibly photoisomerizable fragment ("molecular photoswitch") as part of their structure.…”

“…Among several known photoswitches [18] that are being used in peptides [19][20][21][22][23], diarylethenes (DAE) have increasingly attracted attention in recent years [22,23]. Photoswitchable DAEderived molecules offer several advantages for medical applications, as their photoforms are thermally irreversible and highly fatigue resistant [24][25][26].…”

“…The compound numbers are shown next to the data points. Values for the parent peptide 1 are shown as black filled circles; the original prototype 2 is color-coded in black; the data points for peptides of series i(3)(4)(5) are shown in grey, series ii (6-9) in blue, series iii(10)(11)(12)(13)(14)(15) in red, series iv(16)(17)(18) in green, and series v(19,20) in purple.…”