Flavin Adenine Dinucleotide Chromophore Charge Controls the Conformation of Cyclobutane Pyrimidine Dimer Photolyase α-Helices

Abstract

Observations of light-receptive enzyme complexes are usually complicated by simultaneous overlapping signals from the chromophore, apoprotein, and substrate, so that only the initial, ultrafast, photon-chromophore reaction and the final, slow, protein conformational change provide separate, nonoverlapping signals. Each provides its own advantages, whereas sometimes the overlapping signals from the intervening time scales still cannot be fully deconvoluted. We overcome the problem by using a novel method to selectively isotope-label the apoprotein but not the flavin adenine dinucleotide (FAD) cofactor. This allowed the Fourier transform infrared (FTIR) signals to be separated from the apoprotein, FAD cofactor, and DNA substrate. Consequently, a comprehensive structure-function study by FTIR spectroscopy of the Escherichia coli cyclobutane pyrimidine dimer photolyase (CPD-PHR) DNA repair enzyme was possible. FTIR signals could be identified and assigned upon FAD photoactivation and DNA repair, which revealed protein dynamics for both processes beyond simple one-electron reduction and ejection, respectively. The FTIR data suggest that the synergistic cofactor-protein partnership in CPD-PHR linked to changes in the shape of FAD upon one-electron reduction may be coordinated with conformational changes in the apoprotein, allowing it to fit the DNA substrate. Activation of the CPD-PHR chromophore primes the apoprotein for subsequent DNA repair, suggesting that CPD-PHR is not simply an electron-ejecting structure. When FAD is activated, changes in its structure may trigger coordinated conformational changes in the apoprotein and thymine carbonyl of the substrate, highlighting the role of Glu275. In contrast, during DNA repair and release processes, primary conformational changes occur in the enzyme and DNA substrate, with little contribution from the FAD cofactor and surrounding amino acid residues.

Light‐Induced Conformational Changes in the Plant Cryptochrome Photolyase Homology Region Resolved by Selective Isotope Labeling and Infrared Spectroscopy

“…This assignment is supported by prominent contributions in the amide II range (Fig. b), which are much more pronounced than those observed in CPD photolyase . Previous investigations have only shown very limited changes by steady‐state experiments .…”

“…To date, most conformational changes in cryptochromes upon illumination have been identified or discussed to occur in the CCT . The investigation of possible conformational changes in the PHR of cryptochromes is complementary to a recent focus on such changes in the homologous CPD and (6‐4) photolyases . There, changes in α ‐helices accompany the so‐called photoactivation reaction, in which the fully reduced state is replenished as the starting state for the light‐induced catalysis.…”

“…Thereby, flavin at natural isotope abundance is incorporated during expression (Fig. ), which has been demonstrated to lead to valuable insight by infrared spectroscopy on the mechanism of BLUF (blue light sensor using flavin) domains and CPD photolyases .…”

FTIR study of CPD photolyase with substrate in single strand DNA

“…Our group thus initiated studies of CPD-PHR as well as (6-4) PHR by using light-induced difference FTIR spectroscopy, for which measuring conditions, including sample preparation method, were established [ 8 – 10 ]. The resulting difference spectra indicated structural alterations to FAD, side-chains and the peptide backbone of the enzyme, as well as the substrate [ 8 – 13 ]. In the case of CPD-PHR, we recently reported a comprehensive analysis of isotope labeled 13 C and 15 N using a special E. coli strain, resulting in the successful separation of FAD, substrate and enzyme vibrational signals [ 13 ].…”

“…The resulting difference spectra indicated structural alterations to FAD, side-chains and the peptide backbone of the enzyme, as well as the substrate [ 8 – 13 ]. In the case of CPD-PHR, we recently reported a comprehensive analysis of isotope labeled 13 C and 15 N using a special E. coli strain, resulting in the successful separation of FAD, substrate and enzyme vibrational signals [ 13 ].…”