Hole-burning spectroscopy, a high-resolution spectroscopic technique, allows
details of heterogeneous nano-environments in biological systems to be obtained from
broad absorption bands. Recently, this technique has been applied to proteins,
nucleic acids, cells, and substructures of water to probe the electrostatic
conditions created by macromolecules and the surrounding solvent. Starting with the
factors that obscure the homogeneous linewidth of a chromophore within an
inhomogeneously broadened absorption or emission band, we describe properties and
processes in biological systems that are reflected in the measured hole spectra. The
technique also lends itself to the resolution of perturbation experiments, such as
temperature cycling to elucidate energy landscape barriers, applied external
electric fields (Stark effect) to measure net internal electric fields, and applied
hydrostatic pressure to find the volume compressibility of proteins.
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