Although it is widely known that trimethylamine N-oxide (TMAO), an osmolyte used by nature, stabilizes the folded state of proteins, the underlying mechanism of action is not entirely understood. To gain further insight into this important biological phenomenon, we use the C≡N stretching vibration of an unnatural amino acid, p-cyano-phenylalanine, to directly probe how TMAO affects the hydration and conformational dynamics of a model peptide and a small protein. By assessing how the lineshape and spectral diffusion properties of this vibration change with cosolvent conditions, we are able to show that TMAO achieves its protein-stabilizing ability through the combination of (at least) two mechanisms: (i) It decreases the hydrogen bonding ability of water and hence the stability of the unfolded state, and (ii) it acts as a molecular crowder, as suggested by a recent computational study, that can increase the stability of the folded state via the excluded volume effect.N ature employs a variety of small organic molecules to cope with osmotic stress. Trimethylamine N-oxide (TMAO) is one such naturally occurring osmolyte that protects intracellular components against disruptive stress conditions (1). In particular, previous studies have shown that TMAO is able to enhance protein stability and to counteract the denaturing effect of urea (2, 3). TMAO (Fig. 1, Inset) adopts a skewed tetrahedral structure with a charged oxygen capable of accepting hydrogen bonds (H bonds) and three hydrophobic (methyl) groups. This amphiphilic structural arrangement makes TMAO a rather special cosolvent, because it can form H bonds with water, self-associate in a manner similar to surfactants, and show preferential interactions with or exclusion (4-12) from certain protein functional groups (13-23). Indeed, these molecular properties of TMAO have been used, either individually or in combination, to rationalize its biological activities. For example, a prevailing view about TMAO is that its osmotic and protective role is caused by the molecule's tendency to be preferentially depleted from protein surfaces, as suggested by physicochemical measurements (24-28). This thermodynamic picture, as described by Bolen and coworkers (29), implies that the protein is preferentially hydrated. Although this notion is consistent with TMAO's being a protecting osmolyte, to the best of our knowledge no experimental studies have been carried out to directly examine the effect of TMAO on protein hydration dynamics, an aspect that is also important to protein function. Herein, we use twodimensional infrared (2D IR) spectroscopy and a site-specific IR probe to explore this critical issue and gain insight toward achieving a microscopic understanding of the molecular mechanism of the protecting action of TMAO.2D IR spectroscopy is capable of assessing the frequencyfrequency correlation function (FFCF) of a given IR probe, which reports on the underlying dynamics of events that lead to fluctuations of its vibrational frequency (30). For an IR probe that is able ...
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.
The ability to quantify the local electrostatic environment of proteins and protein/peptide assemblies is key to yielding a microscopic understanding of many biological interactions and processes. Herein, we show that the ester carbonyl stretching vibration of two non-natural amino acids, L-aspartic acid 4-methyl ester and L-glutamic acid 5-methyl ester, is a convenient and sensitive probe in this regard since its frequency correlates linearly with the local electrostatic field for both hydrogen-bonding and non-hydrogen-bonding environments. We expect that the resultant frequency-electric field map will find use in various applications. In addition, we show that, when situated in a non-hydrogen bonding environment, this probe can also be used to measure the local dielectric constant (ε). For example, applying it to amyloid fibrils formed by Aβ16-22 reveals that the interior of such β-sheet assemblies has a ε of ~5.6.
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