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Hydrogen bonding of tryptophan radicals revealed by EPR at 700 GHz.

ABSTRACT: Redox-active tryptophans are important in biological electron transfer and redox biochemistry. Proteins can tune the electron transfer kinetics and redox potentials of tryptophan via control of the protonation state and the hydrogen-bond strength. We examine the local environment of two neutral tryptophan radicals (Trp108 on the solvent-exposed surface and Trp48 buried in the hydrophobic core) in two azurin variants. Ultrahigh-field EPR spectroscopy at 700 GHz and 25 T allowed complete resolution of all of the principal components of the g tensors of the two radicals and revealed significant differences in the g tensor anisotropies. The spectra together with (2)H ENDOR spectra and supporting DFT calculations show that the g tensor anisotropy is directly diagnostic of the presence or absence as well as the strength of a hydrogen bond to the indole nitrogen. The approach is a powerful one for identifying and characterizing hydrogen bonds that are critical in the regulation of tryptophan-assisted electron transfer and tryptophan-mediated redox chemistry in proteins.

SUBMITTER: Stoll S

PROVIDER: S-EPMC3251908 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Hydrogen bonding of tryptophan radicals revealed by EPR at 700 GHz.

Stoll Stefan S Shafaat Hannah S HS Krzystek J J Ozarowski Andrew A Tauber Michael J MJ Kim Judy E JE Britt R David RD

Journal of the American Chemical Society 20111025 45

Redox-active tryptophans are important in biological electron transfer and redox biochemistry. Proteins can tune the electron transfer kinetics and redox potentials of tryptophan via control of the protonation state and the hydrogen-bond strength. We examine the local environment of two neutral tryptophan radicals (Trp108 on the solvent-exposed surface and Trp48 buried in the hydrophobic core) in two azurin variants. Ultrahigh-field EPR spectroscopy at 700 GHz and 25 T allowed complete resolutio ...[more]

PMID: 22007694

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Project description:Ultraviolet resonance Raman (UVRR) spectra of tryptophan compounds in various solvents and a model peptide are presented and reveal systematic changes that reflect solvent polarity, hydrogen bond strength, and cation-pi interaction. The commonly utilized UVRR spectral marker for environment polarity that has been based on off-resonance Raman data, the tryptophan Fermi doublet ratio I1360/I1340, exhibits different values in on- and off-resonance Raman spectra as well as for different tryptophan derivatives. Specifically, the UVRR Fermi doublet ratio for indole ranges from 0.3 in polar solvents to 0.8 in nonpolar solvents, whereas the respective values reported here and previously for off-resonance Raman spectra are 0.5-1.3. UVRR Fermi doublet ratios for the more biologically relevant molecule, N-acetyl tryptophan ethyl ester (NATEE), are in a smaller range of 1.1 (polar solvent) to 1.7 (nonpolar solvent) and correlate to the solvent polarity/polarization parameters pi* and ETN. As has been reported previously, several UVRR modes are also sensitive to the hydrogen bond strength of the indole N-H moiety. Here, we report a new unambiguous marker for H-bonding: the ratio of the W10 (approximately 1237 cm-1) intensity to that of the W9 (approximately 1254 cm-1) mode (RW10). This ratio is 0.7 for NATEE in the absence of hydrogen bond acceptors and increases to 3.1 in the presence of strong hydrogen bond acceptors, with a value of 2.3 in water. The W8 and W17 modes shift more than +10 and approximately -5 cm-1 upon increase in hydrogen bond strength; this range for W17 is smaller than that reported previously and reflects a more realistic range for proteins and peptides in solution. Finally, our data provide evidence for change in the W18 and W16 relative intensity in the presence of cation-pi interactions. These UVRR markers are utilized to interpret spectra of model membrane-bound systems tryptophan octyl ester and the peptide toxin melittin. These spectra reveal the importance of intra- and intermolecular hydrogen bonding and cation-pi interactions that likely influence the partitioning of membrane-associated biomolecules to lipid bilayers or self-associated soluble oligomers. The UVRR analysis presented here modifies and augments prior reports and provides an unambiguous set of spectral makers that can be applied to elucidate the molecular microenvironment and structure of a wide range of complex systems, including anchoring tryptophan residues in membrane proteins and peptides.

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Hydrogen bonding of tryptophan radicals revealed by EPR at 700 GHz.

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Hydrogen bonding of tryptophan radicals revealed by EPR at 700 GHz.

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