13 research outputs found
Functional dynamics of a single tryptophan residue in a BLUF protein revealed by fluorescence spectroscopy
Blue Light Using Flavin (BLUF) domains are increasingly being adopted for use in optogenetic constructs. Despite this, much remains to be resolved on the mechanism of their activation. The advent of unnatural amino acid mutagenesis opens up a new toolbox for the study of protein structural dynamics. The tryptophan analogue, 7-aza-Trp (7AW) was incorporated in the BLUF domain of the Activation of Photopigment and pucA (AppA) photoreceptor in order to investigate the functional dynamics of the crucial W104 residue during photoactivation of the protein. The 7-aza modification to Trp makes selective excitation possible using 310 nm excitation and 380 nm emission, separating the signals of interest from other Trp and Tyr residues. We used Förster energy transfer (FRET) between 7AW and the flavin to estimate the distance between Trp and flavin in both the light- and dark-adapted states in solution. Nanosecond fluorescence anisotropy decay and picosecond fluorescence lifetime measurements for the flavin revealed a rather dynamic picture for the tryptophan residue. In the dark-adapted state, the major population of W104 is pointing away from the flavin and can move freely, in contrast to previous results reported in the literature. Upon blue-light excitation, the dominant tryptophan population is reorganized, moves closer to the flavin occupying a rigidly bound state participating in the hydrogen-bond network around the flavin molecule
Site Specific Protein Dynamics Probed by Ultrafast Infrared Spectroscopy of a Noncanonical Amino Acid
Real-time observation of structure change associated with protein function remains a major challenge. Ultrafast pump-probe methods record dynamics in light activated proteins, but the assignment of spectroscopic observables to specific structure changes can be difficult. The BLUF (blue light using flavin) domain proteins are an important class of light sensing flavoprotein. Here we incorporate the unnatural amino acid (UAA) azidophenylalanine (AzPhe) at key positions in the H-bonding environment of the isoalloxazine chromophore of two BLUF domains, PixD and AppABLUF; both proteins retain the red shift on irradiation characteristic of photoactivity. Steady state and ultrafast time resolved infrared (TRIR) difference measurements of the azido mode reveal site-specific information on the nature and dynamics of light driven structure change. AzPhe dynamics are thus shown to be an effective probe of BLUF domain photoactivation, revealing significant differences between the two proteins, and a differential response of the two sites to chromophore excitation
Variation in LOV Photoreceptor Activation Dynamics Probed by Time-Resolved Infrared Spectroscopy
The light, oxygen, voltage (LOV) domain proteins are blue light photoreceptors that utilize a non-covalently bound flavin mononucleotide (FMN) cofactor as the chromophore. The modular nature of these proteins has led to their wide adoption in the emerging fields of optogenetics and optobiology, where the LOV domain has been fused to a variety of output domains leading to novel light-controlled applications. In the present work, we extend our studies of the sub-picosecond to sub-millisecond transient infrared spectroscopy of the isolated LOV domain AsLOV2 to three full-length photoreceptors in which the LOV domain is fused to an output domain: the LOV-STAS protein, YtvA, the LOV-HTH transcription factor, EL222, and the LOV-histidine kinase, LovK. Despite differences in tertiary structure, the overall pathway leading to cysteine adduct formation from the FMN triplet state is highly conserved, although there are slight variations in rate. However significant differences are observed in the vibrational spectra and kinetics after adduct formation, which are directly linked to the specific output function of the LOV domain. While the rate of adduct formation varies by only 3.6-fold amongst the proteins, the subsequent large-scale structural changes in the full-length LOV photoreceptors occur over the micro- to sub-millisecond timescales and vary by orders of magnitude depending on the different output function of each LOV domain
Unraveling the photoactivation mechanism of a light activated adenylyl cyclase using ultrafast spectroscopy coupled with unnatural amino acid mutagenesis
The hydrogen bonding network that surrounds the flavin in Blue Light Utilizing FAD (BLUF) photoreceptors plays a crucial role in sensing and communicating the changes in the electronic structure of the flavin to the protein matrix upon light absorption. The network contains a highly conserved tyrosine that is essential for photoactivation. Using time-resolved infrared spectroscopy (TRIR) and unnatural amino acid (UAA) incorporation, we investigated the photoactivation mechanism and the role of the conserved tyrosine (Y6) in the forward reaction of the photoactivated adenylyl cyclase (AC) from Oscillatoria Acuminata (OaPAC). Our work elucidates the direct connection between the photoactivation process in the BLUF domain and the structural and functional implications on the partner protein for the first time. The TRIR results demonstrate formation of FADHâ as an intermediate species on the photoactivation pathway which decays to form the signaling state. Using fluorotyrosine analogs to modulate the physical properties of Y6, the TRIR data reveal that a change in the pKa and/or reduction potential of Y6 has a profound effect on the forward reaction, consistent with a mechanism involving proton transfer or proton-coupled electron transfer from Y6 to the electronically excited FAD. Decreasing the pKa from 9.9 to <7.2 and/or increasing the reduction potential by 200 mV of Y6 prevents proton transfer to the flavin and halts the photocycle at FADâ ̶. The lack of protonation of the anionic flavin radical can be directly linked to photoactivation of the AC domain. While the 3F-Y6 and 2,3-F2Y6 variants undergo the complete photocycle and catalyze the conversion of ATP to cAMP, enzyme activity is abolished in the 3,5-F2Y6 and 2,3,5-F3Y6 variants where the photocycle is halted at FADâ ̶. Our results thus show that proton transfer plays an essential role in initiating the structural reorganization of the AC domain that results in adenylyl cyclase activity
Identification of the vibrational marker of tyrosine cation radical using ultrafast transient infrared spectroscopy of flavoprotein systems
Tryptophan and tyrosine radical intermediates play crucial roles in many biological charge transfer processes. Particularly in flavoprotein photochemistry, short-lived reaction intermediates can be studied by the complementary techniques of ultrafast visible and infrared spectroscopy. The spectral properties of tryptophan radical are well established, and the formation of neutral tyrosine radicals has been observed in many biological processes. However, only recently, the formation of a cation tyrosine radical was observed by transient visible spectroscopy in a few systems. Here, we assigned the infrared vibrational markers of the cationic and neutral tyrosine radical at 1483 and 1502 cmâ1 (in deuterated buffer), respectively, in a variant of the bacterial methyl transferase TrmFO, and in the native glucose oxidase. In addition, we studied a mutant of AppABLUF blue-light sensor domain from Rhodobacter sphaeroides in which only a direct formation of the neutral radical was observed. Our studies highlight the exquisite sensitivity of transient infrared spectroscopy to low concentrations of specific radicals
Photocycle alteration and increased enzymatic activity in genetically modified photoactivated adenylate cyclase OaPAC
Photoactivated adenylate cyclases (PACs) are light activated enzymes that combine blue light sensing capacity with the ability to convert ATP to cAMP and pyrophosphate (PPi) in a light-dependent manner. In most of the known PACs blue light regulation is provided by a blue light sensing domain using flavin which undergoes a structural reorganization after blue-light absorption. This minor structural change then is translated toward the C-terminal of the protein, inducing a larger conformational change that results in the ATP conversion to cAMP. As cAMP is a key second messenger in numerous signal transduction pathways regulating various cellular functions, PACs are of great interest in optogenetic studies. The optimal optogenetic device must be âsilentâ in the dark and highly responsive upon light illumination. PAC from Oscillatoria acuminata is a very good candidate as its basal activity is very small in the dark and the conversion rates increase 20-fold upon light illumination. We studied the effect of replacing D67 to N, in the blue light using flavin domain. This mutation was found to accelerate the primary electron transfer process in the photosensing domain of the protein, as has been predicted. Furthermore, it resulted in a longer lived signaling state, which was formed with a lower quantum yield. Our studies show that the overall effects of the D67N mutation lead to a slightly higher conversion of ATP to cAMP, which points in the direction that by fine tuning the kinetic properties more responsive PACs and optogenetic devices can be generated
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Unraveling the Mechanism of a LOV Domain Optogenetic Sensor:A Glutamine Lever Induces Unfolding of the Jα Helix
Light-activated protein domains provide a convenient, modular, and genetically encodable sensor for optogenetics and optobiology. Although these domains have now been deployed in numerous systems, the precise mechanism of photoactivation and the accompanying structural dynamics that modulate output domain activity remain to be fully elucidated. In the C-terminal light-oxygen-voltage (LOV) domain of plant phototropins (LOV2), blue light activation leads to formation of an adduct between a conserved Cys residue and the embedded FMN chromophore, rotation of a conserved Gln (Q513), and unfolding of a helix (Jα-helix) which is coupled to the output domain. In the present work, we focus on the allosteric pathways leading to Jα helix unfolding in Avena sativa LOV2 (AsLOV2) using an interdisciplinary approach involving molecular dynamics simulations extending to 7 ÎŒs, time-resolved infrared spectroscopy, solution NMR spectroscopy, and in-cell optogenetic experiments. In the dark state, the side chain of N414 is hydrogen bonded to the backbone N-H of Q513. The simulations predict a lever-like motion of Q513 after Cys adduct formation resulting in a loss of the interaction between the side chain of N414 and the backbone CâO of Q513, and formation of a transient hydrogen bond between the Q513 and N414 side chains. The central role of N414 in signal transduction was evaluated by site-directed mutagenesis supporting a direct link between Jα helix unfolding dynamics and the cellular function of the Zdk2-AsLOV2 optogenetic construct. Through this multifaceted approach, we show that Q513 and N414 are critical mediators of protein structural dynamics, linking the ultrafast (sub-ps) excitation of the FMN chromophore to the microsecond conformational changes that result in photoreceptor activation and biological function