Formation of a New Buried Charge Drives a Large-Amplitude Protein Quake in Photoreceptor Activation

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We present a comprehensive study that integrates experimental and theoretical nonequilibrium techniques to map energy landscapes along well defined pull-axis specific coordinates to elucidate mechanisms of protein unfolding. Single-molecule force-extension experiments along two different axes of photoactive yellow protein combined with nonequilibrium statistical mechanical analysis and atomistic simulation reveal energetic and mechanistic anisotropy. Steered molecular dynamics simulations and free-energy curves constructed from the experimental results reveal that unfolding along one axis exhibits a transition-state-like feature where six hydrogen bonds break simultaneously with weak interactions observed during further unfolding. The other axis exhibits a constant (unpeaked) force profile indicative of a noncooperative transition, with enthalpic (e.g., H-bond) interactions being broken throughout the unfolding process. Striking qualitative agreement was found between the force-extension curves derived from steered molecular dynamics calculations and the equilibrium freeenergy curves obtained by Jarzynski-Hummer-Szabo analysis of the nonequilibrium work data. The anisotropy persists beyond pulling distances of more than twice the initial dimensions of the folded protein, indicating a rich energy landscape to the mechanically fully unfolded state. Our findings challenge the notion that cooperative unfolding is a universal feature in protein stability.nonequilibrium dynamics ͉ photoactive yellow protein ͉ biophysics A key step toward connecting protein structure, dynamics, and function is the insightful mapping of energy landscapes (1). A proper set of reaction coordinates encodes the progress of a transition through the dynamical bottleneck region and correlates energetics with structure. Significant advances, both experimental and computational, have been made to map energy landscapes and understand protein (un)folding mechanisms (2). Experimental methods such as fluorescence quenching (3), fluorescence resonance energy transfer (4), hydrogen exchange (5), and small-angle x-ray scattering (6) yielded insights into the rates and structures of folding intermediates. A common result emerging from these studies on a range of small water-soluble proteins is that protein unfolding occurs in a cooperative, all-or-none, transition with a single dominant barrier separating the native and unfolded states (7,8). However, these approaches tend to sample thermodynamically favorable pathways and do not address why other paths are not preferred. For example, a gradual progression of partially unfolded intermediates is usually not detected for these small proteins, possibly because such conformational states are not populated to a measurable extent. Recently, thermal unfolding studies exhibited thermodynamically noncooperative behavior (9, 10). Hence, the molecular basis for the cooperativity of protein folding is a matter of considerable debate (11).In addition, unfolding (i.e., both chemical and mechanical) is a highly nonequ...

Section: Background On the Photoactive Yellow Protein Systemmentioning

confidence: 63%

Section: Background On the Photoactive Yellow Protein Systemmentioning

confidence: 99%

Axis-dependent anisotropy in protein unfolding from integrated nonequilibrium single-molecule experiments, analysis, and simulation

Nome

Zhao

Hoff

et al. 2007

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“…S1B and Table S5) only eight are located at the photoactive site of PYP (24). Of these residues, only Tyr42, Glu46, and Cys69 play a classical role as active site residues in that they serve as hydrogen bonding partners (20), proton donor (39), and cofactor attachment site (23). Fifteen highly conserved side chains are far removed from the active site.…”

Section: Discussionmentioning

confidence: 99%

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

Philip

Kumauchi

Hoff

2010

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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

“…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%

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

Zhao

Lee²,

Nome

et al. 2006

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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...