Toward an Alternative Intrinsic Probe for Spectroscopic Characterization of a Protein

Abstract

The intrinsic fluorescent amino acid tryptophan is the unanimous choice for the spectroscopic investigation of proteins. However, several complicacies in the interpretation of tryptophan fluorescence in a protein are inevitable and an alternative intrinsic protein probe is a longstanding demand. In this contribution, we report an electron-transfer reaction in a human transporter protein (HSA) cavity which causes the tryptophan residue (Trp214) to undergo chemical modification to form one of its metabolites kynurenine (Kyn214). Structural integrity upon modification of the native protein is confirmed by dynamic light scattering (DLS) as well as near and far circular dichroism (CD) spectroscopy. Femtosecond-resolved fluorescence transients of the modified protein describe the dynamics of solvent molecules in the protein cavity in both the native and denatured states. In order to establish general use of the probe, we have studied the dipolar interaction of Kyn214 with a surface-bound ligand (crystal violet, CV) of the protein. By using the sensitivity of FRET, we have determined the distance between Kyn214 (donor) and CV (acceptor). Our study is an attempt to explore an alternative intrinsic fluorescence probe for the spectroscopic investigation of a protein. In order to establish the efficacy of the modification technique we have converted the tryptophan residues of other proteins (bovine serum albumin, chymotrypsin and subtilisin Carlsberg) to kynurenine and confirmed their structural integrity. We have also shown that catalytic activity of the enzymes remains intact upon the modification.

Oxidation Enhances Human Serum Albumin Thermal Stability and Changes the Routes of Amyloid Fibril Formation

“…kynurenine (Kyn214). This would lead to significant variations in the absorption and fluorescence spectrum [58]. However, no significant changes were measured in the absorption spectrum of the two samples, which are perfectly superimposable (data not reported).…”

A Potential Carcinogenic Pyrene Derivative under Förster Resonance Energy Transfer to Various Energy Acceptors in Nanoscopic Environments

“…The refractive index, n , of the medium was measured as 1.3, which is apparent, because the space separating the donor and the acceptor consists of the hydrophobic tails (alkyl chain) of the SDS micelle and the refractive index of similar alkanes such as pentane, hexane, heptane, dodecane, and so on varies between 1.30 and 1.42. The quantum yield, Q D , of the donor in the absence of the acceptor was calculated according to Equation (3):32 …”

Slow Solvent Relaxation Dynamics of Nanometer Sized Reverse Micellar Systems Through Tryptophan Metabolite, Kynurenine

“…OD and OD R are the optical densities of KN in the RM of different w 0 values and reference at the excitation wavelength 375 nm and n and n R are the refractive indices of KN in the RM of different w 0 values and reference solutions respectively. The absolute quantum yield of protein bound KN (1) was taken to be 4.8 × 10 −3 . Refractive indices of the solutions were measured by using Rudolph J357 automatic refractometer.…”

“… Abstract Exploration of environmental dynamics using intrinsic biological probe tryptophan is very important; however, it suffers from various difficulties. An alternative probe, kynurenine (KN), has been found to be an efficient probe for the ultrafast dynamics in the biological environment (Goswami et al. , [2010] J. Phys.…”