Photochemical tools to study dynamic biological processes

Abstract

Light-responsive biologically active compounds offer the possibility to study the dynamics of biological processes. Phototriggers and photoswitches have been designed, providing the capability to rapidly cause the initiation of wide range of dynamic biological phenomena. We will discuss, in this article, recent developments in the field of light-triggered chemical tools, specially how two-photon excitation, "caged" fluorophores, and the photoregulation of protein activities in combination with time-resolved x-ray techniques should break new grounds in the understanding of dynamic biological processes.

Figure 1. Photoregulation of functional proteins using a phototrigger or a photoswitch.

Figure 2. Several photolabile groups used for the caging of biologically active compounds (LG=leaving group).

Figure 3. (a) Two-photon versus one-photon processes.

(b) Fluorescence localization in one-photon (all light pathway) and two-photon (focal point) excitation of an organic fluorophore (Hayek et al., 2006) (c) Dissymmetric donor-acceptor (CDNI-Glu) and symmetric acceptor-acceptor (BNSF-Glu) caged glutamates with high two-photon uncaging action cross sections.

Figure 4. Cellular protein trafficking studies using caged fluorophores and small genetically encoded peptide tags.

Figure 5. Proposed mechanism for the photoregulation of transporter protein activity using photocleavable crosslinkers.

This class of membrane proteins needs to undergo an important structural rearrangement to catalyze the transport of specific molecules or ions across membrane bilayer. Therefore, they could be locked in one conformation by using both site-directed double cysteine mutants of the protein and intramolecular crosslinking. The UV photolysis of such modified transporters should rapidly and efficiently cleave the crosslinker, leading to the triggering of their activities.