Role of an N-Terminal Loop in the Secondary Structural Change of Photoactive Yellow Protein

Harigai, Miki; Imamoto, Yasushi; Kamikubo, Hironari; Yamazaki, Yoichi; Kataoka, Mikio

doi:10.1021/bi034814e

Cited by 60 publications

(133 citation statements)

References 38 publications

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“…6). Thus, the experimental and computational results reported here demonstrate that light-triggered destabilization and partial unfolding of PYP occur in its core PAS domain, in contrast to the previous bulk solution measurements indicating unfolding of only the N-terminal ␣-helices of PYP (13)(14)(15).…”

Section: Force-extension Curves In the Dark And Under Illuminationcontrasting

confidence: 77%

“…The emerging mechanism of receptor activation in PYP contains the following steps: (i) initial chromophore photoisomerization (32), which triggers (ii) active site proton transfer (33) that causes (iii) an electrostatically triggered protein quake (12), resulting in structural disruption of the PAS domain core, which is relayed (iv) to the N-terminal helices (13,14) by the side chain of Asn-43 (34). Future studies are needed to reveal how partial PAS domain unfolding contributes to signaling and whether this mechanism operates more widely in the PAS domain family.…”

Section: Discussionmentioning

confidence: 99%

“…Thus, the degree to which different regions of PYP unfold in solution upon pB formation remains an open question. Previous solution spectroscopy experiments indicate that unfolding of the two N-terminal helices of PYP is involved (10,(13)(14)(15). Recent NMR measurements on a PYP mutant lacking the 25 N-terminal residues indicate that regions outside the central ␤-sheet also undergo a significant increase in flexibility (16).…”

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confidence: 99%

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Single-molecule detection of structural changes during Per-Arnt-Sim (PAS) domain activation

Zhao

Lee²,

Nome

et al. 2006

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

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The Per-Arnt-Sim (PAS) domain is a ubiquitous protein module with a common three-dimensional fold involved in a wide range of regulatory and sensory functions in all domains of life. The activation of these functions is thought to involve partial unfolding of N-or C-terminal helices attached to the PAS domain. Here we use atomic force microscopy to probe receptor activation in single molecules of photoactive yellow protein (PYP), a prototype of the PAS domain family. Mechanical unfolding of Cys-linked PYP multimers in the presence and absence of illumination reveals that, in contrast to previous studies, the PAS domain itself is extended by Ϸ3 nm (at the 10-pN detection limit of the measurement) and destabilized by Ϸ30% in the light-activated state of PYP. Comparative measurements and steered molecular dynamics simulations of two double-Cys PYP mutants that probe different regions of the PAS domain quantify the anisotropy in stability and changes in local structure, thereby demonstrating the partial unfolding of their PAS domain upon activation. These results establish a generally applicable single-molecule approach for mapping functional conformational changes to selected regions of a protein. In addition, the results have profound implications for the molecular mechanism of PAS domain activation and indicate that stimulusinduced partial protein unfolding can be used as a signaling mechanism.atomic force spectroscopy ͉ photoactive yellow protein ͉ receptor activation B iological signaling starts with the activation of receptor proteins, in which stimuli trigger protein conformational changes that relay a signal to an associated signal transduction chain. A central question concerns the nature of the structural changes that activate a receptor protein. The mechanism of signaling by PerArnt-Sim (PAS) domain-containing proteins is of considerable interest, because these structural domains occur in many signaling proteins (1). In addition, incorrect signaling by PAS domains is associated with a range of diseases. Important examples are mutations in the human ERG potassium channel, which causes cardiac arrhythmias (2), and the involvement of hypoxia-inducible factors in myocardial and cerebral ischemia (3).We investigate the photoinduced structural changes in the bacterial blue light receptor, photoactive yellow protein (PYP), a prototype for PAS domains (4). As shown in Fig. 1, PYP consists of two N-terminal ␣-helices (residues 1-25), followed by a typical PAS domain (1, 5) fold (residues 26-125) that contains a central six-stranded ␤-sheet flanked by ␣-helices (6). The protein exhibits photochemistry based on its p-coumaric acid chromophore (7). Blue light converts the initial pG state of PYP into the photoactivated pB state in milliseconds, whereas pB decays back to pG in Ϸ0.5 s (8). The pB state is believed to trigger negative phototaxis in purple bacteria (9).The structural changes that occur upon pB formation involve partial unfolding of the receptor, indicating partial protein unfolding as a novel signaling m...

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Section: Force-extension Curves In the Dark And Under Illuminationcontrasting

confidence: 77%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Single-molecule detection of structural changes during Per-Arnt-Sim (PAS) domain activation

Zhao

Lee²,

Nome

et al. 2006

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

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“…Note that the pB 0 state reported here does not have the same global structure as that produced in solution, where the N-terminal domain is free to move beyond the constraints imposed by crystal packing. In our kinetic model, the crystalinaccessible "signaling" intermediate, in which the N-terminal domain is believed to be partially unfolded (28)(29)(30), is denoted pB 1 .…”

Section: Discussionmentioning

confidence: 99%

Watching a signaling protein function in real time via 100-ps time-resolved Laue crystallography

Schotte

Cho

Kaila

et al. 2012

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

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179

244

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To understand how signaling proteins function, it is crucial to know the time-ordered sequence of events that lead to the signaling state. We recently developed on the BioCARS 14-IDB beamline at the Advanced Photon Source the infrastructure required to characterize structural changes in protein crystals with near-atomic spatial resolution and 150-ps time resolution, and have used this capability to track the reversible photocycle of photoactive yellow protein (PYP) following trans-to-cis photoisomerization of its p-coumaric acid (pCA) chromophore over 10 decades of time. The first of four major intermediates characterized in this study is highly contorted, with the pCA carbonyl rotated nearly 90°out of the plane of the phenolate. A hydrogen bond between the pCA carbonyl and the Cys69 backbone constrains the chromophore in this unusual twisted conformation. Density functional theory calculations confirm that this structure is chemically plausible and corresponds to a strained cis intermediate. This unique structure is short-lived (∼600 ps), has not been observed in prior cryocrystallography experiments, and is the progenitor of intermediates characterized in previous nanosecond time-resolved Laue crystallography studies. The structural transitions unveiled during the PYP photocycle include trans/cis isomerization, the breaking and making of hydrogen bonds, formation/ relaxation of strain, and gated water penetration into the interior of the protein. This mechanistically detailed, near-atomic resolution description of the complete PYP photocycle provides a framework for understanding signal transduction in proteins, and for assessing and validating theoretical/computational approaches in protein biophysics.time-resolved X-ray diffraction | photoreceptor | light sensor

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“…Significant inhibition was observed around 1 M of NaCl (Fig. 3(c)) suggesting that two co-refolded enzymes might have weaker ionic interactions between N-and C-terminal fragments than the native enzyme [14,15].…”

Section: Effect Of Ph and Temperature On Enzyme Activitymentioning

confidence: 99%

Co‐refolding of two peptide fragments derived from Agrobacterium tumefaciens β‐glucosidase with catalytic activity

2005

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Four sites of the non-homologous region (coding amino acid residues of 347, 421, 466 and 533) of a gene were randomly selected for splitting to investigate the function of b-glucosidase from Agrobacterium tumefaciens in the co-refolding of peptides into the catalytically active enzyme. As a result of gene splitting, four N-and C-terminal domain peptides were obtained as insoluble inclusion bodies. No catalytic activity was observed when these fragments refolded individually. However, a considerable amount of activity was restored when the two fragments derived from N-and C-terminal peptides were co-refolded together. The deletion of amino acid residues in the non-homologous region resulted in a complete loss of enzyme activity, which suggests that truncation of amino acids in this region strongly affects the co-refolding ability of the enzyme to maintain activity.

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Role of an N-Terminal Loop in the Secondary Structural Change of Photoactive Yellow Protein

Cited by 60 publications

References 38 publications

Single-molecule detection of structural changes during Per-Arnt-Sim (PAS) domain activation

Single-molecule detection of structural changes during Per-Arnt-Sim (PAS) domain activation

Watching a signaling protein function in real time via 100-ps time-resolved Laue crystallography

Co‐refolding of two peptide fragments derived from Agrobacterium tumefaciens β‐glucosidase with catalytic activity

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