Vibrational spectroscopy provides a direct route to the physicochemical characterization of molecules. While both IR and Raman spectroscopy have been used for decades to provide detailed characterizations of small molecules, similar studies with proteins are largely precluded due to spectral congestion. However, the vibrational spectra of proteins do include a "transparent window", between ∼1800 and ∼2500 cm, and progress is now being made to develop site-specifically incorporated carbon-deuterium (C-D), cyano (CN), thiocyanate (SCN), and azide (N) "transparent window vibrational probes" that absorb within this window and report on their environment to facilitate the characterization of proteins with small molecule-like detail. This Review opens with a brief discussion of the advantages and limitations of conventional vibrational spectroscopy and then discusses the strengths and weaknesses of the different transparent window vibrational probes, methods by which they may be site-specifically incorporated into peptides and proteins, and the physicochemical properties they may be used to study, including electrostatics, stability and folding, hydrogen bonding, protonation, solvation, dynamics, and interactions with inhibitors. The use of the probes to vibrationally image proteins and other biomolecules within cells is also discussed. We then present four case studies, focused on ketosteroid isomerase, the SH3 domain, dihydrofolate reductase, and cytochrome c, where the transparent window vibrational probes have already been used to elucidate important aspects of protein structure and function. The Review concludes by highlighting the current challenges and future potential of using transparent window vibrational probes to understand the evolution and function of proteins and other biomolecules.

The evolution of proteins with novel function is thought to start from precursor proteins that are conformationally heterogeneous. The corresponding genes may be duplicated and then mutated to select and optimize a specific conformation. However, testing this idea has been difficult because of the challenge of quantifying protein flexibility and conformational heterogeneity as a function of evolution. Here, we report the characterization of protein heterogeneity and dynamics as a function of evolution for the antifluorescein antibody 4-4-20. Using nonlinear laser spectroscopy, surface plasmon resonance, and molecular dynamics simulations, we demonstrate that evolution localized the Ab-combining site from a heterogeneous ensemble of conformations to a single conformation by introducing mutations that act cooperatively and over significant distances to rigidify the protein. This study demonstrates how protein dynamics may be tailored by evolution and has important implications for our understanding of how novel protein functions are evolved.flexibility ͉ nonlinear spectroscopy ͉ fluorscein ͉ molecular recognition

Probe probation: The cyano group is sensitive to its environment, absorbs in a unique region of protein IR spectra, and may be appended to an amino acid. When investigated in variants of cytochrome c (see picture: heme‐pocket structure) by steady‐state and time‐resolved methods, it was found to be a useful site‐specific probe of protein microenvironments and dynamics; however, it can also perturb its environment and destabilize the folded state of the protein.

The alkaline-induced structural transitions of ferricytochrome c have been studied intensively as a model for how changes in metal ligation contribute to protein function and folding. Previous studies have demonstrated that multiple non-native species accumulate with increasing pH. Here, we used a combination of experiments and simulations to provide a high-resolution view of the changes associated with increasing alkaline conditions. Alkaline-induced transitions were characterized under equilibrium conditions by following changes in the IR absorptions of carbon-deuterium chromophores incorporated at Leu68, Lys72, Lys73, Lys79, and Met80. The data suggest that at least four intermediates are formed as the pH is increased prior to complete unfolding of the protein. The first alkaline transition observed appears to be driven by a single deprotonation and occurs with a midpoint of pH 8.8, but surprisingly, the intermediate formed does not appear to be one of the wellcharacterized lysine misligates. At higher pH, second and third deprotonations, with a combined apparent midpoint pH of 10.2, induce transitions to Lys73-or Lys79-misligated species. Interestingly, the lysine misligates appear to undergo iron reduction by the coordinated amine. A transition from the lysine misligates to another intermediate, likely a hydroxide-misligated species, is associated with a fourth deprotonation and a midpoint of pH 10.7. Finally, the protein loses tertiary structure with a fifth deprotonation that occurs with a midpoint of pH 12.7. Native topology-based models with enforced misligation are employed to help understand the structures of the observed intermediates.The energy landscape underlying the folding and function of ferricytochrome c (cyt c) 1 is dominated by the covalently attached heme cofactor and its coordination chemistry (2 , 3). In fact, misligation of the heme center with different protein ligands was one of the first examples of frustration on an energy landscape (4). Ligation and misligation of cyt c have been † This work was supported by the National Science Foundation under Grant MCB 0346967. Any opinions, findings, and conclusions expressed here are those of the author(s) and do not necessarily reflect the views of the National Science Foundation. C-D carbon-deuterium FT IRFourier-transform infrared. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 April 12. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript intensively studied since 1941, when Theorell and Åkesson first reported that the oxidized protein populates at least five distinct states between pH 1 and pH 13, denoted I to V ( 5). State III is the native form of the protein with Met80 bound to the heme center, and it dominates under neutral conditions. At higher pH, multiple misligated species accumulate, including a lysine-misligated state IV and a hydroxide-misligated state V ,2 before the protein eventually unfolds (3,6,7). The transitions between the various states have serve...