Methods to probe protein transitions with ATR infrared spectroscopy.

We describe techniques that can be used in conjunction with modern attenuated total reflection (ATR) infrared micro-prisms to allow proteins to be manipulated cyclically between different states whilst simultaneously monitoring both mid-IR and UV/visible/near IR changes. These methods provide increased flexibility of the types of changes that can be induced in proteins in comparison to transmission methods. Quantitative measurements can be made of vibrational changes associated with conversion between stable catalytic reaction intermediates, ligand binding and oxidation-reduction. Both hydrophobic and soluble proteins can be analysed and the ability to induce transitions repetitively allows IR difference spectra to be acquired at a signal/noise sufficient to resolve changes due to specific cofactors or amino acids. Such spectra can often be interpreted at the atomic level by standard IR methods of comparisons with model compounds, by isotope and mutation effects and, increasingly, by ab initio simulations. Combination of such analyses with atomic 3D structural models derived from X-ray and NMR studies can lead to a deeper understanding of molecular mechanisms of enzymatic reactions.