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...

Octaanionic meso-tetra(3,5-dicarboxylatophenyl) porphyrin 1 was adsorbed to gold electrodes at pH 12 and stayed there after repeated washing with 10-2 M KOH. The fluorescence on sputtered gold surfaces amounted to 10% of the intensity observed on an organic subphase. Addition of 10-6 M aqueous solutions of the manganese(III) complexes of an isomer mixture of tetracationic β-tetraethyl-β‘-tetrakis(1-methyl-4-pyridinium)- and meso-4-(1-methyl-4-pyridinium)phenyl porphyrins 2 and 4 at pH 12 quenched the fluorescence quantitatively. Visible spectroscopy proved that the amount of porphyrin 1 on the gold surface had not changed. The octaanionic porphyrin 1 was then embedded in a membrane by self-assembly of a bolaamphiphile containing two secondary amide groups. Two hydrogen bond chains rigidify such a monolayer. The emission of porphyrin 1 remained after the self-assembly process. 1 was now localized on the bottom of a rigid membrane gap. Its fluorescence was again quantitatively quenched by the tetracationic manganese(III) porphyrinate 2, which fit in with the membrane gap. A larger manganese(III) porphyrin with a phenyl spacer between the porphyrin and methyl pyridinium rings could not enter, and no quenching was observed. The same experiment with a more fluid membrane made of octadecanethiol showed no such discriminating effect. The entrapment of 1,2-trans-cyclohexanediol within the “immobile” water volume of the membrane gap is also reported. Water-soluble compounds have thus been separated within a 2 nm3 water volume from bulk water. So far, the membrane pores with a porphyrin bottom resemble natural enzyme clefts.

The production of antibodies that selectively bind virtually any foreign compound is the hallmark of the immune system. While much is understood about how sequence diversity contributes to this remarkable feat of molecular recognition, little is known about how sequence diversity impacts antibody dynamics, which is also expected to contribute to molecular recognition. Toward this goal, we examined a panel of antibodies elicited to the chromophoric antigen fluorescein. On the basis of isothermal titration calorimetry, we selected six antibodies that bind fluorescein with diverse binding entropies, suggestive of varying contributions of dynamics to molecular recognition. Sequencing revealed that two pairs of antibodies employ homologous heavy chains that were derived from common germline genes, while the other two heavy chains and all six of the light chains were derived from different germline genes and are not homologous. Interestingly, more than half of all the somatic mutations acquired during affinity maturation among the six antibodies are located in positions unlikely to contact fluorescein directly. To quantify and compare the dynamics of the antibody-fluorescein complexes, three-pulse photon echo peak shift and transient grating spectroscopy were employed. All of the antibodies exhibited motions on three distinct time scales, ultrafast motions on the <100 fs time scale, diffusive motions on the picosecond time scale, and motions that occur on time scales longer than nanoseconds and thus appear static. However, the exact frequency of the picosecond time scale motion and the relative contribution of the different motions vary significantly among the antibody-chromophore complexes, revealing a high level of dynamic diversity. Using a hierarchical model, we relate the data to features of the antibodies' energy landscapes as well as their flexibility in terms of elasticity and plasticity. In all, the data provide a consistent picture of antibody flexibility, which interestingly appears to be correlated with binding entropy as well as with germline gene use and the mutations introduced during affinity maturation. The data also provide a gauge of the dynamic diversity of the antibody repertoire and suggest that this diversity might contribute to molecular recognition by facilitating the recognition of the broadest range of foreign molecules.

A variety of IR-active moieties with absorptions that are distinct from those of proteins have been developed as probes of local protein environments, including carbon-deuterium bonds (CD), cyano groups (CN), and azides (N3 ); however, no systematic analysis of their utility in a protein has been published. Previously, we characterized the N-terminal Src homology 3 domain of the murine adapter protein Crk-II (nSH3) with CD bonds site-selectively incorporated throughout, and showed that it is relatively rigid and electrostatically heterogeneous and that it thermally unfolds under equilibrium conditions via a simple two-state mechanism. We now report the synthesis and characterization of eight variants of nSH3 with CN and/or N3 probes at five of the same positions. In agreement with previous studies, the position-dependent spectra suggest that both probes are predominantly sensitive to hydration, and not to their local electrostatic environments. Importantly, both probes also tend to significantly perturb the protein if they are not incorporated at surface-exposed positions. Thus, unlike CD labels, which are both sensitive to their environment and non-perturbative, CN and N3 probes should be used with caution.

The covalently bound heme cofactor plays a dominant role in the folding of cytochrome c. Due to the complicated inorganic chemistry of the heme, some might consider the folding of cytochrome c to be a special case that follows different principles than those used to describe folding of proteins without cofactors. Recent investigations, however, demonstrate that models which are commonly used to describe folding for many proteins work well for cytochrome c when heme is explicitly introduced and generally provide results that agree with experimental observations. We will first discuss results from simple native structure-based models. These models include attractive interactions between nonadjacent residues only if they are present in the crystal structure at pH 7. Since attractive nonnative contacts are not included in native structure-based models, their energy landscapes can be described as “perfectly funneled.” In other words, native structure-based models are energetically guided towards the native state and contain no energetic traps that would hinder folding. Energetic traps are sources of frustration which cause specific transient intermediates to be populated. Native structure-based models do include repulsion between residues due to excluded volume. Nonenergetic traps can therefore exist if the chain, which cannot cross over itself, must partially unfold in order for folding to proceed. The ability of native structure-based models to capture these type of motions is in part responsible for their successful predictions of folding pathways for many types of proteins. Models without frustration describe well the sequence of folding events for cytochrome c inferred from hydrogen exchange experiments thereby justifying their use as a starting point. At low pH, the folding sequence of cytochrome c deviates from that at pH 7 and from those predicted from models with perfectly funneled energy landscapes. Alternate folding pathways are a result of “chemical frustration.” This frustration arises because some regions of the protein are destabilized more than others due to the heterogeneous distribution of titratable residues that are protonated at low pH. We construct more complex models that include chemical frustration, in addition to the native structure-based terms. These more complex models only modestly perturb the energy landscape which remains overall well funneled. These perturbed models can accurately describe how alternative folding pathways are used at low pH. At alkaline pH, cytochrome c populates distinctly different structural ensembles. For instance, lysine residues are deprotonated and compete for the heme ligation site. The same models that can describe folding at low pH also predict well the structures and relative stabilities of intermediates populated at alkaline pH.