Excited State Structure and Dynamics of the Neutral and Anionic Flavin Radical Revealed by Ultrafast Transient Mid-IR to Visible Spectroscopy

Neutral and anionic flavin radicals are involved in numerous photochemical processes and play an essential part in forming the signaling state of various photoactive flavoproteins such as cryptochromes and BLUF domain proteins. A stable neutral radical flavin has been prepared for study in aqueous solution, and both neutral and anion radical states have been stabilized in the proteins flavodoxin and glucose oxidase. Ultrafast transient absorption measurements were performed in the visible and mid-infrared region in order to characterize the excited state dynamics and the excited and ground state vibrational spectra and to probe the effect of the protein matrix on them. These data are compared with the results of density functional theory calculations. Excited state decay dynamics were found to be a strong function of the protein matrix. The ultrafast electron transfer quenching mechanism of the excited flavin moiety in glucose oxidase is characterized by vibrational spectroscopy. Such data will be critical in the ongoing analysis of the photocycle of photoactive flavoproteins

Proteins in Action: Femtosecond to Millisecond Structural Dynamics of a Photoactive Flavoprotein

Living systems are fundamentally dependent on the ability of proteins to respond to external stimuli. The mechanism, the underlying structural dynamics, and the time scales for regulation of this response are central questions in biochemistry. Here we probe the structural dynamics of the BLUF domain found in several photoactive flavoproteins, which is responsible for light activated functions as diverse as phototaxis and gene regulation. Measurements have been made over 10 decades of time (from 100 fs to 1 ms) using transient vibrational spectroscopy. Chromophore (flavin ring) localized dynamics occur on the pico- to nanosecond time scale, while subsequent protein structural reorganization is observed over microseconds. Multiple time scales are observed for the dynamics associated with different vibrations of the protein, suggesting an underlying hierarchical relaxation pathway. Structural evolution in residues directly H-bonded to the chromophore takes place more slowly than changes in more remote residues. However, a point mutation which suppresses biological function is shown to ‘short circuit’ this structural relaxation pathway, suppressing the changes which occur further away from the chromophore while accelerating dynamics close to it

Probing the Complex Binding Modes of the PPARγ Partial Agonist 2‑Chloro‑<i>N</i>‑(3-chloro-4-((5-chlorobenzo[<i>d</i>]thiazol-2-yl)thio)phenyl)-4-(trifluoromethyl)benzenesulfonamide (T2384) to Orthosteric and Allosteric Sites with NMR Spectroscopy

In a previous study, a cocrystal structure of PPARγ bound to 2-chloro-<i>N</i>-(3-chloro-4-((5-chlorobenzo­[<i>d</i>]­thiazol-2-yl)­thio)­phenyl)-4-(trifluoromethyl)­benzenesulfonamide (<b>1</b>, T2384) revealed two orthosteric pocket binding modes attributed to a concentration-dependent biochemical activity profile. However, <b>1</b> also bound an alternate/allosteric site that could alternatively account for the profile. Here, we show ligand aggregation afflicts the activity profile of <b>1</b> in biochemical assays. However, ligand-observed fluorine (¹⁹F) and protein-observed NMR confirms <b>1</b> binds PPARγ with two orthosteric binding modes and to an allosteric site

Vibrational Assignment of the Ultrafast Infrared Spectrum of the Photoactivatable Flavoprotein AppA

The blue light using flavin (BLUF) domain proteins, such as the transcriptional antirepressor AppA, are a novel class of photosensors that bind flavin noncovalently in order to sense and respond to high-intensity blue (450 nm) light. Importantly, the noncovalently bound flavin chromophore is unable to undergo large-scale structural change upon light absorption, and thus there is significant interest in understanding how the BLUF protein matrix senses and responds to flavin photoexcitation. Light absorption is proposed to result in alterations in the hydrogen-bonding network that surrounds the flavin chromophore on an ultrafast time scale, and the structural changes caused by photoexcitation are being probed by vibrational spectroscopy. Here we report ultrafast time-resolved infrared spectra of the AppA BLUF domain (AppA_BLUF) reconstituted with isotopes of FAD, specifically [U-¹³C₁₇]-FAD, [xylene-¹³C₈]-FAD, [U-¹⁵N₄]-FAD, and [4-¹⁸O₁]-FAD both in solution and bound to AppA_BLUF. This allows for unambiguous assignment of ground- and excited-state modes arising directly from the flavin. Studies of model compounds and DFT calculations of the ground-state vibrational spectra reveal the sensitivity of these modes to their environment, indicating they can be used as probes of structural dynamics

Identification of a Binding Site for Unsaturated Fatty Acids in the Orphan Nuclear Receptor Nurr1

Nurr1/NR4A2 is an orphan nuclear receptor, and currently there are no known natural ligands that bind Nurr1. A recent metabolomics study identified unsaturated fatty acids, including arachidonic acid and docosahexaenoic acid (DHA), that interact with the ligand-binding domain (LBD) of a related orphan receptor, Nur77/NR4A1. However, the binding location and whether these ligands bind other NR4A receptors were not defined. Here, we show that unsaturated fatty acids also interact with the Nurr1 LBD, and solution NMR spectroscopy reveals the binding epitope of DHA at its putative ligand-binding pocket. Biochemical assays reveal that DHA-bound Nurr1 interacts with high affinity with a peptide derived from PIASγ, a protein that interacts with Nurr1 in cellular extracts, and DHA also affects cellular Nurr1 transactivation. This work is the first structural report of a natural ligand binding to a canonical NR4A ligand-binding pocket and indicates a natural ligand can bind and affect Nurr1 function

Mechanism of the AppA_BLUF Photocycle Probed by Site-Specific Incorporation of Fluorotyrosine Residues: Effect of the Y21 p<i>K</i>_a on the Forward and Reverse Ground-State Reactions

The transcriptional antirepressor AppA is a blue light using flavin (BLUF) photoreceptor that releases the transcriptional repressor PpsR upon photoexcitation. Light activation of AppA involves changes in a hydrogen-bonding network that surrounds the flavin chromophore on the nanosecond time scale, while the dark state of AppA is then recovered in a light-independent reaction with a dramatically longer half-life of 15 min. Residue Y21, a component of the hydrogen-bonding network, is known to be essential for photoactivity. Here, we directly explore the effect of the Y21 p<i>K</i>_a on dark state recovery by replacing Y21 with fluorotyrosine analogues that increase the acidity of Y21 by 3.5 pH units. Ultrafast transient infrared measurements confirm that the structure of AppA is unperturbed by fluorotyrosine substitution, and that there is a small (3-fold) change in the photokinetics of the forward reaction over the fluorotyrosine series. However, reduction of 3.5 pH units in the p<i>K</i>_a of Y21 increases the rate of dark state recovery by 4000-fold with a Brønsted coefficient of ∼1, indicating that the Y21 proton is completely transferred in the transition state leading from light to dark adapted AppA. A large solvent isotope effect of ∼6–8 is also observed on the rate of dark state recovery. These data establish that the acidity of Y21 is a crucial factor for stabilizing the light activated form of the protein, and have been used to propose a model for dark state recovery that will ultimately prove useful for tuning the properties of BLUF photosensors for optogenetic applications