Using Nitrile-Derivatized Amino Acids as Infrared Probes of Local Environment

Getahun, Zelleka; Huang, Cheng‐Yen; Wang, Ting; León, Brenda De; DeGrado, William F.; Gai, Feng

doi:10.1021/ja0285262

Cited by 330 publications

(571 citation statements)

References 26 publications

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“…In comparison, these linear IR results indicate that TPA + has a larger effect on the local environment of the C≡N probe than Gdm + . For both Phe CN and Trp CN , upon changing the solvent from water to tetrahydrofuran (THF) the C≡N stretching vibrational band is shifted to a lower frequency and also becomes narrower (28,36). Therefore, these linear IR results further suggest that (i) both TPA + and Gdm + (to a lesser extent) can affect the local hydration status of aromatic side chains and (ii) TPA + , but not Gdm + , can specifically accumulate around those aromatic moieties because the latter does not lead to any significant increase in the IR bandwidth and hence the inhomogeneous part of the C≡N vibrational linewidth.…”

Section: Resultsmentioning

confidence: 99%

Do guanidinium and tetrapropylammonium ions specifically interact with aromatic amino acid side chains?

Ding

Mukherjee

Chen

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Many ions are known to affect the activity, stability, and structural integrity of proteins. Although this effect can be generally attributed to ion-induced changes in forces that govern protein folding, delineating the underlying mechanism of action still remains challenging because it requires assessment of all relevant interactions, such as ion-protein, ion-water, and ion-ion interactions. Herein, we use two unnatural aromatic amino acids and several spectroscopic techniques to examine whether guanidinium chloride, one of the most commonly used protein denaturants, and tetrapropylammonium chloride can specifically interact with aromatic side chains. Our results show that tetrapropylammonium, but not guanidinium, can preferentially accumulate around aromatic residues and that tetrapropylammonium undergoes a transition at ∼1.3 M to form aggregates. We find that similar to ionic micelles, on one hand, such aggregates can disrupt native hydrophobic interactions, and on the other hand, they can promote α-helix formation in certain peptides.T he stability of a protein, or more precisely, the free energy difference between its folded and unfolded states, can be modulated by various solution properties. For example, addition of another solute to a protein solution can result in either a decrease or an increase in this protein's stability (1-3). The most noticeable example in this regard is the Hofmeister series (4-6), a group of ions that are ranked based on their protein-denaturing abilities. Among this series, the guanidinium ion (Gdm + ) is the most widely used chemical agent in protein denaturation due to its strong destabilizing effect and high solubility in water. Consequently, its mechanism of action has been subjected to extensive studies (7)(8)(9)(10)(11)(12)(13)(14). Although different interpretations have been put forth (15-17), the generally accepted notion is that Gdm + denatures a protein by preferentially interacting with its peptide groups (7,11,18), including certain side chains (11,(19)(20)(21)(22). In particular, it has been hypothesized that Gdm + , which exists in aqueous solution as a rigid, flat object (12,18,19), can engage in stacking interactions with amino acids consisting of planar side chains, such as arginine (Arg) (19), asparagine (Asn) (11,20), glutamine (Gln) (11,20), and aromatic residues (20)(21)(22). However, to the best of our knowledge, the only experimental evidence that supports this hypothesis comes from crystallographic data (20)(21)(22), which show that the guanidinium group of Arg is more frequently found to be stacked against the side chain of tyrosine (Tyr) or tryptophan (Trp) in proteins. Therefore, additional experimental studies that can directly probe such stack interactions in solution are needed.Another ion in the Hofmeister series that has a similar proteindenaturing capability to Gdm + is tetrapropylammonium (TPA + ) (23). However, a recent study by Dempsey et al. (24) found that although TPA + , like Gdm + , can denature a tryptophan (Trp) zipper β-hairpin (i.e....

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Section: Resultsmentioning

confidence: 99%

Do guanidinium and tetrapropylammonium ions specifically interact with aromatic amino acid side chains?

Ding

Mukherjee

Chen

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…32,33 Cyano groups absorb in a region of the IR spectrum that is free of protein absorptions (~2200 cm −1 ), which facilitates both their detection and analysis. In addition, they may be attached to amino acids and incorporated into different parts of a protein.…”

Section: Introductionmentioning

confidence: 99%

“…These interactions are not native to the protein but are likely to dominate the probe's absorption properties. 32,34 An alternative approach that does not rely on the introduction of artificial probes is based on the characterization of native protein absorptions using isotope labeling and difference Fourier transform infrared (FT IR) spectroscopy. This method has been widely used to examine heavy atom vibrations, such as those associated with amide bonds.…”

Section: Introductionmentioning

confidence: 99%

Carbon−Deuterium Bonds as Probes of Dihydrofolate Reductase

2008

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Much effort has been directed towards understanding the contributions of electrostatics and dynamics to protein function and especially to enzyme catalysis. Unfortunately, these studies have been limited by the absence of direct experimental probes. We have been developing the use of carbon-deuterium bonds as probes of proteins and now report the application of the technique to the enzyme dihydrofolate reductase, which catalyzes a hydride transfer and has served as a paradigm for biological catalysis. We observe that the stretching absorption frequency of (methyl-d 3 ) methionine carbon-deuterium bonds show an approximately linear dependence on solvent dielectric. Solvent and computational studies support the empirical interpretation of the stretching frequency in terms of local polarity. To begin to explore the use of this technique to study enzyme function and mechanism, we report a preliminary analysis of (methyl-d 3 ) methionine residues within dihydrofolate reductase. Specifically, we characterize the IR absorptions at Met16 and Met20, within the catalytically important Met20 loop, and Met42, which is located within the hydrophobic core of the enzyme. The results confirm the sensitivity of the carbon-deuterium bonds to their local protein environment, demonstrate that dihydrofolate reductase is electrostatically and dynamically heterogeneous, and lay the foundation for the direct characterization protein electrostatics and dynamics, and potentially, their contribution to catalysis.

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“…For example, this approach has been used to identify the existence of mobile water molecules inside Aβ40 amyloid fibrils (31,32) and to interrogate the water-assisted drug-binding mechanism of HIV-1 reverse transcriptase (33), among many other applications (34)(35)(36)(37)(38)(39). In the current study, we capitalize on the established sensitivity of the nitrile stretching vibration (C≡N) to local hydration and electrostatic environment (40) and use the unnatural amino acid p-cyano-phenyalanine (Phe CN ) as a local IR reporter. The advantages of using the C≡N stretching vibration of Phe CN are that (i) it is located in a spectrally uncongested region (2,000−2,400 cm −1 ), where water has a relatively low absorbance; (ii) it has a reasonably large extinction coefficient; and (iii) it is, in most cases, a simple transition that is decoupled from other vibrational modes of the molecule (41).…”

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confidence: 99%

Microscopic insights into the protein-stabilizing effect of trimethylamine N-oxide (TMAO)

Pazos

Gai

2014

Proc. Natl. Acad. Sci. U.S.A.

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237

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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 ...

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Using Nitrile-Derivatized Amino Acids as Infrared Probes of Local Environment

Cited by 330 publications

References 26 publications

Do guanidinium and tetrapropylammonium ions specifically interact with aromatic amino acid side chains?

Do guanidinium and tetrapropylammonium ions specifically interact with aromatic amino acid side chains?

Carbon−Deuterium Bonds as Probes of Dihydrofolate Reductase

Microscopic insights into the protein-stabilizing effect of trimethylamine N-oxide (TMAO)

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