Characterization of Red/Green Cyanobacteriochrome NpR6012g4 by Solution Nuclear Magnetic Resonance Spectroscopy: A Hydrophobic Pocket for the C15-E,anti Chromophore in the Photoproduct.

Cyanobacteriochromes (CBCRs) are cyanobacterial photosensory proteins distantly related to phytochromes. Like phytochromes, CBCRs reversibly photoconvert between a dark-stable state and a photoproduct via photoisomerization of the 15,16-double bond of their linear tetrapyrrole (bilin) chromophores. CBCRs provide cyanobacteria with complete coverage of the visible spectrum and near-ultraviolet region. One CBCR subfamily, the canonical red/green CBCRs typified by AnPixJg2 and NpR6012g4, can function as sensors of light color or intensity because of their great variation in photoproduct stability. The mechanistic basis for detection of green light by the photoproduct state in this subfamily has proven to be a challenging research topic, with competing hydration and trapped-twist models proposed. Here, we use ¹³C-edited and ¹5N-edited ¹H-¹H NOESY solution nuclear magnetic resonance spectroscopy to probe changes in chromophore configuration and protein-chromophore interactions in the NpR6012g4 photocycle. Our results confirm a C15-Z,anti configuration for the red-absorbing dark state and reveal a C15-E,anti configuration for the green-absorbing photoproduct. The photoactive chromophore D-ring is located in a hydrophobic environment in the photoproduct, surrounded by both aliphatic and aromatic residues. Characterization of variant proteins demonstrates that no aliphatic residue is essential for photoproduct tuning. Taken together, our results support the trapped-twist model over the hydration model for the red/green photocycle of NpR6012g4.

Comparison of effect of gamma ray irradiation on wild-type and N-terminal mutants of αA-crystallin.

PURPOSE: To study the comparative structural and functional changes between wild-type (wt) and N-terminal congenital cataract causing αA-crystallin mutants (R12C, R21L, R49C, and R54C) upon exposure to different dosages of gamma rays. METHODS: Alpha A crystallin N-terminal mutants were created with the site-directed mutagenesis method. The recombinantly overexpressed and purified wt and mutant proteins were used for further studies. A (60)Co source was used to generate gamma rays to irradiate wild and mutant proteins at dosages of 0.5, 1.0, and 2.0 kGy. The biophysical property of the gamma irradiated (GI) and non-gamma irradiated (NGI) αA-crystallin wt and N-terminal mutants were determined. Oligomeric size was determined by size exclusion high-performance liquid chromatography (HPLC), the secondary structure with circular dichroism (CD) spectrometry, conformation of proteins with surface hydrophobicity, and the functional characterization were determined regarding chaperone activity using the alcohol dehydrogenase (ADH) aggregation assay. RESULTS: αA-crystallin N-terminal mutants formed high molecular weight (HMW) cross-linked products as well as aggregates when exposed to GI compared to the NGI wt counterparts. Furthermore, all mutants exhibited changed ß-sheet and random coil structure. The GI mutants demonstrated decreased surface hydrophobicity when compared to αA-crystallin wt at 0, 1.0, and 1.5 kGy; however, at 2.0 kGy a drastic increase in hydrophobicity was observed only in the mutant R54C, not the wt. In contrast, chaperone activity toward ADH was gradually elevated at the minimum level in all GI mutants, and significant elevation was observed in the R12C mutant. CONCLUSIONS: Our findings suggest that the N-terminal mutants of αA-crystallin are structurally and functionally more sensitive to GI when compared to their NGI counterparts and wt. Protein oxidation as a result of gamma irradiation drives the protein to cross-link and aggregate culminating in cataract formation.

Identification and affinity-quantification of ß-amyloid and α-synuclein polypeptides using on-line SAW-biosensor-mass spectrometry.

Bioaffinity analysis using a variety of biosensors has become an established tool for detection and quantification of biomolecular interactions. Biosensors, however, are generally limited by the lack of chemical structure information of affinity-bound ligands. On-line bioaffinity-mass spectrometry using a surface-acoustic wave biosensor (SAW-MS) is a new combination providing the simultaneous affinity detection, quantification, and mass spectrometric structural characterization of ligands. We describe here an on-line SAW-MS combination for direct identification and affinity determination, using a new interface for MS of the affinity-isolated ligand eluate. Key element of the SAW-MS combination is a microfluidic interface that integrates affinity-isolation on a gold chip, in-situ sample concentration, and desalting with a microcolumn for MS of the ligand eluate from the biosensor. Suitable MS-acquisition software has been developed that provides coupling of the SAW-MS interface to a Bruker Daltonics ion trap-MS, FTICR-MS, and Waters Synapt-QTOF- MS systems. Applications are presented for mass spectrometric identifications and affinity (K(D)) determinations of the neurodegenerative polypeptides, ß-amyloid (Aß), and pathophysiological and physiological synucleins (α- and ß-synucleins), two key polypeptide systems for Alzheimer's disease and Parkinson's disease, respectively. Moreover, first in vivo applications of αSyn polypeptides from brain homogenate show the feasibility of on-line affinity-MS to the direct analysis of biological material. These results demonstrate on-line SAW-bioaffinity-MS as a powerful tool for structural and quantitative analysis of biopolymer interactions.

An active photoreceptor intermediate revealed by in situ photoirradiated solid-state NMR spectroscopy.

A novel, to our knowledge, in situ photoirradiation system for solid-state NMR measurements is improved and demonstrated to successfully identify the M-photointermediate of pharaonis phoborhodopsin (ppR or sensory rhodopsin II), that of the complex with transducer (ppR/pHtrII), and T204A mutant embedded in a model membrane. The (13)C NMR signals from [20-(13)C]retinal-ppR and ppR/pHtrII revealed that multiple M-intermediates with 13-cis, 15-anti retinal configuration coexisted under the continuously photoirradiated condition. NMR signals observed from the photoactivated retinal provide insights into the process of photocycle in the ppR/pHtrII complex.

Light-induced conformational changes in full-length Arabidopsis thaliana cryptochrome.

Cryptochromes (CRYs) are widespread flavoproteins with homology to photolyases (PHRs), a class of blue-light-activated DNA repair enzymes. Unlike PHRs, both plant and animal CRYs have a C-terminal domain. This cryptochrome C-terminal (CCT) domain mediates interactions with other proteins, while the PHR-like domain converts light energy into a signal via reduction and radical formation of the flavin adenine dinucleotide cofactor. However, the mechanism by which the PHR-like domain regulates the CCT domain is not known. Here, we applied the pulsed-laser-induced transient grating method to detect conformational changes induced by blue-light excitation of full-length Arabidopsis thaliana cryptochrome 1 (AtCRY1). A significant reduction in the diffusion coefficient of AtCRY1 was observed upon photoexcitation, indicating that a large conformational change occurs in this monomeric protein. AtCRY1 containing a single mutation (W324F) that abolishes an intra-protein electron transfer cascade did not exhibit this conformational change. Moreover, the conformational change was much reduced in protein lacking the CCT domain. Thus, we conclude that the observed large conformational changes triggered by light excitation of the PHR-like domain result from C-terminal domain rearrangement. This inter-domain modulation would be critical for CRYs' ability to transduce a blue-light signal into altered protein-protein interactions for biological activity. Lastly, we demonstrate that the transient grating technique provides a powerful method for the direct observation and understanding of photoreceptor dynamics.

Analysis of red-fluorescent proteins provides insight into dark-state conversion and photodegradation.

Fluorescent proteins (FPs) are powerful tools that permit real-time visualization of cellular processes. The utility of a given FP for a specific experiment depends strongly on its effective brightness and overall photostability. However, the brightness of FPs is limited by dark-state conversion (DSC) and irreversible photobleaching, which occur on different timescales. Here, we present in vivo ensemble assays for measuring DSC and irreversible photobleaching under continuous and pulsed illumination. An analysis of closely related red FPs reveals that DSC and irreversible photobleaching are not always connected by the same mechanistic pathway. DSC occurs out of the first-excited singlet state, and its magnitude depends predominantly on the kinetics for recovery out of the dark state. The experimental results can be replicated through kinetic simulations of a four-state model of the electronic states. The methodology presented here allows light-driven dynamics to be studied at the ensemble level over six orders of magnitude in time (microsecond to second timescales).

Kinetics of conformational changes of the FKF1-LOV domain upon photoexcitation.

The photochemical reaction dynamics of a light-oxygen-voltage (LOV) domain from the blue light sensor protein, FKF1 (flavin-binding Kelch repeat F-box) was studied by means of the pulsed laser-induced transient grating method. The observed absorption spectral changes upon photoexcitation were similar to the spectral changes observed for typical LOV domain proteins (e.g., phototropins). The adduct formation took place with a time constant of 6 µs. After this reaction, a significant conformational change with a time constant of 6 ms was observed as a change in the diffusion coefficient. An FKF1-LOV mutant without the conserved loop connecting helices E and F, which is present only in the FKF1/LOV Kelch protein 2/ZEITLUPE family, did not show these slow phase dynamics. This result indicates that the conformational change in the loop region represents a major change in the FKF1-LOV photoreaction.