Transient Exposure of Hydrophobic Surface in the Photoactive Yellow Protein Monitored with Nile Red

Hendriks, Johnny; Gensch, Thomas; Hviid, Lene; Horst, Michael; Hellingwerf, Klaas J.; Thor, Jasper J. van

doi:10.1016/s0006-3495(02)75514-8

Cited by 100 publications

(123 citation statements)

References 40 publications

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“…This conformation exposes Ϸ80 Å 2 of additional solvent accessible surface area relative to E-PYP (Fig. 6), an area sufficient to bind Nile Red, a hydrophobic dye, to a region adjacent to the chromophore in the I 2 state of E-PYP (18). Furthermore, a hydrogen bond, which is present in ground state E-PYP between the side chain of Arg-52 and the backbone carbonyl oxygen of residue 98 (Fig.…”

Section: Conformational Substates and Signaling Inmentioning

confidence: 98%

“…After excitation by blue light, the ground state, whose maximal absorption is at 446 nm, converts through a series of red-shifted intermediates I 0 , I 0 ‡ , and I 1 , in picoseconds, hundreds of picoseconds, and nanoseconds respectively (14), before decaying to a blue-shifted intermediate I 2 , which completes the photocycle by reverting to the ground state in hundreds of milliseconds (15). Because it is the longest-lived state in the photocycle, I 2 is considered to be the ''signaling state'' of E-PYP and is associated with structural heterogeneity (12), partial protein unfolding (16), and transient exposure of hydrophobic residues (17,18). The photocycle and its intermediates have been studied by a number of crystallographic (9-11, 19, 20) and spectroscopic methods (12,14,15,(21)(22)(23)(24).…”

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confidence: 99%

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Crystal structure of a photoactive yellow protein from a sensor histidine kinase: Conformational variability and signal transduction

Rajagopal

Moffat

2003

Proc. Natl. Acad. Sci. U.S.A.

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Photoactive yellow protein (E-PYP) is a blue light photoreceptor, implicated in a negative phototactic response in Ectothiorhodospira halophila, that also serves as a model for the Per–Arnt–Sim superfamily of signaling molecules. Because no biological signaling partner for E-PYP has been identified, it has not been possible to correlate any of its photocycle intermediates with a relevant signaling state. However, the PYP domain (Ppr-PYP) from the sensor histidine kinase Ppr in Rhodospirillum centenum, which regulates the catalytic activity of Ppr by blue light absorption, may allow such issues to be addressed. Here we report the crystal structure of Ppr-PYP at 2 Å resolution. This domain has the same absorption spectrum and similar photocycle kinetics as full length Ppr, but a blue-shifted absorbance and considerably slower photocycle than E-PYP. Although the overall fold of Ppr-PYP resembles that of E-PYP, a novel conformation of the β4–β5 loop results in inaccessibility of Met-100, thought to catalyze chromophore reisomerization, to the chromophore. This conformation also exposes a highly conserved molecular surface that could interact with downstream signaling partners. Other structural differences in the α3–α4 and β4–β5 loops are consistent with these regions playing significant roles in the control of photocycle dynamics and, by comparison to other sensory Per–Arnt–Sim domains, in signal transduction. Because of its direct linkage to a measurable biological output, Ppr-PYP serves as an excellent system for understanding how changes in photocycle dynamics affect signaling by PYPs

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Section: Conformational Substates and Signaling Inmentioning

confidence: 98%

mentioning

confidence: 99%

Crystal structure of a photoactive yellow protein from a sensor histidine kinase: Conformational variability and signal transduction

Rajagopal

Moffat

2003

Proc. Natl. Acad. Sci. U.S.A.

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“…This indicates that the absorption spectrum characteristic for the orange form is a result of the specific protein-chromophore interactions provided by a distinct protein conformation. OCP photoconversion leads to reduction of the protein order (21), partial unfolding (17,19,20), or formation of the socalled molten globule state (24), which is typical for some other photoactive proteins (25). By definition, a molten globule has an increased protein volume (26), which was demonstrated for OCP R by small-angle x-ray scattering (SAXS) and size-exclusion chromatography (17,22).…”

Section: Introductionmentioning

confidence: 98%

Fluorescent Labeling Preserving OCP Photoactivity Reveals Its Reorganization during the Photocycle

Maksimov

Sluchanko

Mironov

et al. 2017

Biophysical Journal

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Orange carotenoid protein (OCP), responsible for the photoprotection of the cyanobacterial photosynthetic apparatus under excessive light conditions, undergoes significant rearrangements upon photoconversion and transits from the stable orange to the signaling red state. This is thought to involve a 12-Å translocation of the carotenoid cofactor and separation of the N-and C-terminal protein domains. Despite clear recent progress, the detailed mechanism of the OCP photoconversion and associated photoprotection remains elusive. Here, we labeled the OCP of Synechocystis with tetramethylrhodamine-maleimide (TMR) and obtained a photoactive OCP-TMR complex, the fluorescence of which was highly sensitive to the protein state, showing unprecedented contrast between the orange and red states and reflecting changes in protein conformation and the distances from TMR to the carotenoid throughout the photocycle. The OCP-TMR complex was sensitive to the light intensity, temperature, and viscosity of the solvent. Based on the observed Fö rster resonance energy transfer, we determined that upon photoconversion, the distance between TMR (donor) bound to a cysteine in the C-terminal domain and the carotenoid (acceptor) increased by 18 Å , with simultaneous translocation of the carotenoid into the N-terminal domain. Time-resolved fluorescence anisotropy revealed a significant decrease of the OCP rotation rate in the red state, indicating that the light-triggered conversion of the protein is accompanied by an increase of its hydrodynamic radius. Thus, our results support the idea of significant structural rearrangements of OCP, providing, to our knowledge, new insights into the structural rearrangements of OCP throughout the photocycle and a completely novel approach to the study of its photocycle and non-photochemical quenching. We suggest that this approach can be generally applied to other photoactive proteins.

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“…One important example is the use of the curved temperature dependence of the kinetics of pB decay (27) or the use of fluorescent dyes (37,38) to achieve high-throughput detection of the extent of light-induced conformational changes. More generally, the approach reported here can be applied to any protein for which (i) sufficient amount of protein can be overproduced and purified by Ni-NTA affinity chromatography, and (ii) the properties of the protein can be detected spectroscopically either by an intrinsic chromophore (aromatic side chains or cofactors) or by attached fluorescent probes or chromogenic substrate derivatives.…”

Section: Discussionmentioning

confidence: 99%

Robustness and evolvability in the functional anatomy of a PER-ARNT-SIM (PAS) domain

Philip

Kumauchi

Hoff

2010

Proc. Natl. Acad. Sci. U.S.A.

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The robustness of proteins against point mutations implies that only a small subset of residues determines functional properties. We test this prediction using photoactive yellow protein (PYP), a 125-residue prototype of the PER-ARNT-SIM (PAS) domain superfamily of signaling proteins. PAS domains are defined by a small number of conserved residues of unknown function. We report high-throughput biophysical measurements on a complete Ala scan set of purified PYP mutants. The dataset of 1,193 values on active site properties, functional kinetics, stability, and production level reveals that 124 mutants retain the characteristic photocycle of PYP, but that the majority of substitutions significantly alter functional properties. Only 35% of substitutions that strongly affect function are located at the active site. Unexpectedly, most PAS-conserved residues are required for maintaining protein production. PAS domain activation often involves conformational changes in α-helices linked to the PAS core. However, the mechanism of transmission and kinetic regulation of allosteric structural changes from the PAS domain to these helices is not clear. The Ala scan data reveal interactions governing allosteric switching in PYP. The photocycle kinetics is significantly altered by substitutions at 58 positions and spans a 3,000-fold range. Nine residues that dock the N-terminal α-helices of PYP to its PAS core regulate signaling kinetics. Ile39 and Asn43 are identified as part of a mechanism for regulating allosteric switching that is conserved among PAS domains. These results show that PYP combines robustness with a high degree of evolvability and imply production level as an important factor in protein evolution.allosteric signal transmission | molecular evolution | protein structure-function relationship

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Transient Exposure of Hydrophobic Surface in the Photoactive Yellow Protein Monitored with Nile Red

Cited by 100 publications

References 40 publications

Crystal structure of a photoactive yellow protein from a sensor histidine kinase: Conformational variability and signal transduction

Crystal structure of a photoactive yellow protein from a sensor histidine kinase: Conformational variability and signal transduction

Fluorescent Labeling Preserving OCP Photoactivity Reveals Its Reorganization during the Photocycle

Robustness and evolvability in the functional anatomy of a PER-ARNT-SIM (PAS) domain

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