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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T , respectively. No changes in highresolution static crystallographic T(deoxy)-and R(oxy)-quaternary and tertiary structures of Hb and their heme environment, as well as the axial coordination structures of the deoxy-heme (n Fe-His = 215 /cm ) and the oxy-heme (n Fe-O2 = 567 /cm ) in solution, are observed, despite K T and K R values are changed as much as 100-and 2,000-folds, respectively. Thus, the assumption that the low-affinity state is caused by the inter-dimeric salt-bridge-linked constraints, the out-of-plane shift of the heme Fe, and the allosteric core constraint in the T(deoxy)-Hb is no longer valid. Although these constraints are completely absent in R(oxy)-Hb, its O 2 -affinity is modulated as much as 2,000-folds by its interaction with heterotropic effectors. The effector-linked modulation of thermal fluctuation of the protein may be responsible.

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Free divers can’t hold their breath as long as whales, but they train their bodies to maximize their oxygen (O2) storing potential using the protein myoglobin. Myoglobin’s structure has been known for decades, but researchers are still trying to determine just how myoglobin functions. Found in muscle tissue, myoglobin stores O2, a molecule needed to produce chemical energy. Toxic ligands, such as carbon monoxide (CO) and cyanide, also bind to myoglobin. When CO binds to a free heme group, the heme's binding affinity for CO is 20,000 times that for O2. When heme is surrounded by myoglobin, that binding affinity ratio drops to only 25. The decrease was thought to be due to steric interactions which prevented CO from occupying the same space as His64. Recent evidence suggests that electrostatic interactions and hydrogen bonds play a more important role. The O2 is stabilized as opposed to the CO being pushed out. Several amino acids (His64, Val68, Phe43, Phe46, and Leu29,) seem to stabilize the ligand. With 3D printing technology, the Brown Deer SMART (Students Modeling a Research Topic) Team created a model of myoglobin. If researchers can fully understand ligand discrimination by heme proteins, not only will divers be able to hold their breath longer, but we may be able to cure diseases like anemia where there is a lack of O2 in the blood. Grant Funding Source: The SMART team program is supported by a grant from NIH‐CTSA.