Studying submicrosecond protein folding kinetics using a photolabile caging strategy and time‐resolved photoacoustic calorimetry

Abstract

Kinetic measurement of protein folding is limited by the method used to trigger folding. Traditional methods, such as stopped flow, have a long mixing dead time and cannot be used to monitor fast folding processes. Here, we report a compound, 4-(bromomethyl)-6,7-dimethoxycoumarin, that can be used as a "photolabile cage" to study the early stages of protein folding. The folding process of a protein, RD1, including kinetics, enthalpy, and volume change, was studied by the combined use of a phototriggered caging strategy and time-resolved photoacoustic calorimetry. The cage caused unfolding of the photolabile protein, and then a pulse UV laser (∼10(-9) s) was used to break the cage, leaving the protein free to refold and allowing the resolving of two folding events on a nanosecond time scale. This strategy is especially good for monitoring fast folding proteins that cannot be studied by traditional methods.

Spectroscopic studies of protein folding: Linear and nonlinear methods

“…Another method of conformational switching is to use a relatively large molecular moiety (i.e., a cage) to force the protein of interest to unfold under native conditions; on cleavage of the cage by excitation with an appropriate laser pulse, the protein will fold to its native conformation. Thus, several photocages, such as benzoinyl cages, have been used to trigger protein folding kinetics 39–43. Photocaged molecules can also be used in a cyclization scheme to produce a photocleavable intramolecular linker.…”

Directly monitor protein rearrangement on a nanosecond-to-millisecond time-scale

“…In our previous work on RD1 refolding by the same caging strategy 29 , the photocleavage yield was estimated based on caged peptide due to the similarity between caged RD1 and caged peptide in the solvent accessibility of the cage. However, DMC is shown to be buried inside and quite isolated from water molecules in the case of V5C-DMC.…”

Tightening up the structure, lighting up the pathway: application of molecular constraints and light to manipulate protein folding, self-assembly and function

“…The disadvantage, on the other hand, is that typically more sample is needed and, in some cases, the diffusion of the photoproduct out of the protein matrix can significantly decrease the effective time-resolution of the method. Examples of photocages that have been used to study protein folding include 4,5-dimethoxy-2-nitrobenzene [69] and 4-(bromomethyl)-6,7-dimethoxycoumarin [70]. …”