Carbonyl-based blue autofluorescence of proteins and amino acids.

Chamani Niyangoda,Leonid Breydo,Martin Muschol,Tatiana Miti,Vladimir Uversky,Reza Khodarahmi

doi:10.1371/journal.pone.0176983

Chamani Niyangoda, Leonid Breydo + Show 4 more

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https://doi.org/10.1371/journal.pone.0176983

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Abstract

Intrinsic protein fluorescence is inextricably linked to the near-UV autofluorescence of aromatic amino acids. Here we show that a novel deep-blue autofluorescence (dbAF), previously thought to emerge as a result of protein aggregation, is present at the level of monomeric proteins and even poly- and single amino acids. Just as its aggregation-related counterpart, this autofluorescence does not depend on aromatic residues, can be excited at the long wavelength edge of the UV and emits in the deep blue. Differences in dbAF excitation and emission peaks and intensities from proteins and single amino acids upon changes in solution conditions suggest dbAF’s sensitivity to both the chemical identity and solution environment of amino acids. Autofluorescence comparable to dbAF is emitted by carbonyl-containing organic solvents, but not those lacking the carbonyl group. This implicates the carbonyl double bonds as the likely source for the autofluorescence in all these compounds. Using beta-lactoglobulin and proline, we have measured the molar extinction coefficients and quantum yields for dbAF in the monomeric state. To establish its potential utility in monitoring protein biophysics, we show that dbAF emission undergoes a red-shift comparable in magnitude to tryptophan upon thermal denaturation of lysozyme, and that it is sensitive to quenching by acrylamide. Carbonyl dbAF therefore provides a previously neglected intrinsic optical probe for investigating the structure and dynamics of amino acids, proteins and, by extension, DNA and RNA.

Highlights

Autofluorescence from proteins is typically equated with the intrinsic UV fluorescence of tryptophan and, to a lesser extent, tyrosine and phenylalanine. [1] The molecular substrate for the intrinsic fluorescence in these three amino acids arises from the delocalized electronic states of their indole and phenyl residues, respectively
To establish its potential utility in monitoring protein biophysics, we show that deep-blue autofluorescence (dbAF) emission undergoes a red-shift comparable in magnitude to tryptophan upon thermal denaturation of lysozyme, and that it is sensitive to quenching by acrylamide
To confirm the important role of carbonyls as the structural element underlying this autofluorescence, we looked for intrinsic fluorescence from methanol (H3COH) and isopropanol (CH3)2CHOH, in which the carbonyl in formaldehyde and acetone is replaced by a hydroxyl group

Summary

Introduction

Autofluorescence from proteins is typically equated with the intrinsic UV fluorescence of tryptophan and, to a lesser extent, tyrosine and phenylalanine. [1] The molecular substrate for the intrinsic fluorescence in these three amino acids arises from the delocalized electronic states of their indole and phenyl residues, respectively. Autofluorescence from proteins is typically equated with the intrinsic UV fluorescence of tryptophan and, to a lesser extent, tyrosine and phenylalanine. The molar absorptivity and high quantum yield, combined with the intrinsic sensitivity of tryptophan fluorescence to its environment, has made tryptophan a widely used optical probe for measuring numerous aspects of protein structure and dynamics. Carbonyl-based blue autofluorescence of proteins and amino acids label-free readout of many aspects of protein properties and their sensitivity to the solution environment. Organic compounds that contain carbonyls, such as formaldehyde and acetone, displayed autofluorescence with spectral characteristics similar to dbAF from amino acids and proteins. We propose that carbonyls are the dominant molecular substrate for the observed dbAF from amino acid and proteins

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