Infrared spectroscopy has played an instrumental role in studying a wide variety of biological questions. However, in many cases it is impossible or difficult to rely on the intrinsic vibrational modes of biological molecules of interest, such as proteins, to reveal structural and/or environmental information in a site-specific manner. To overcome this limitation, many recent efforts have been dedicated to the development and application of various extrinsic vibrational probes that can be incorporated into biological molecules and used to site-specifically interrogate their structural and/or environmental properties. In this Review, we highlight some recent advancements of this rapidly growing research area.

Elucidating the underlying molecular mechanisms of protein folding and function is a very exciting and active research area, but poses significant challenges. This is due in part to the fact that existing experimental techniques are incapable of capturing snapshots along the ‘reaction coordinate’ in question with both sufficient spatial and temporal resolutions. In this regard, recent years have seen increased interests and efforts in development and employment of site-specific probes to enhance the structural sensitivity of spectroscopic techniques in conformational and dynamical studies of biological molecules. In particular, the spectroscopic and chemical properties of nitriles, thiocyanates, and azides render these groups attractive for the interrogation of complex biochemical constructs and processes. Here, we review their signatures in vibrational, fluorescence and NMR spectra and their utility in the context of elucidating chemical structure and dynamics of protein and DNA molecules.

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A new twist: A multi‐probe and multi‐frequency approach is shown for dissecting the folding dynamics of individual protein structural elements. In response to a temperature jump the 310‐helix (blue in the picture) of the miniprotein Trp‐cage unfolds before the global unfolding of the protein, whereas the formation of the cage structure depends on the folding of the α‐helix (red).

Amyotrophic lateral sclerosis (ALS) is a devastating fatal syndrome characterized by very rapid degeneration of motor neurons. A leading hypothesis is that ALS is caused by toxic protein misfolding and aggregation, as also occurs in many other neurodegenerative disorders, such as prion, Alzheimer's, Parkinson's, and Huntington's diseases. A prominent cause of familial ALS is mutations in the protein superoxide dismutase (SOD1), which promote the formation of misfolded SOD1 conformers that are prone to aberrant interactions both with each other and with other cellular components. We have shown previously that immature SOD1, lacking bound Cu and Zn metal ions and the intrasubunit disulfide bond (apoSOD1), has a rugged free-energy surface (FES) and exchanges with four other conformations (excited states) that have millisecond lifetimes and sparse populations on the order of a few percent. Here, we examine further states of SOD1 along its maturation pathway, as well as those off-pathway resulting from metal loss that have been observed in proteinaceous inclusions. Metallation and disulfide bond formation lead to structural transformations including local ordering of the electrostatic loop and native dimerization that are observed in rare conformers of apoSOD1; thus, SOD1 maturation may occur via a population-switch mechanism whereby posttranslational modifications select for preexisting structures on the FES. Metallation and oxidation of SOD1 stabilize the native, mature conformation and decrease the number of detected excited conformational states, suggesting that it is the immature forms of the protein that contribute to misfolded conformations in vivo rather than the highly stable enzymatically active dimer.