Kinetic, Energetic, and Mechanical Differences between Dark-State Rhodopsin and Opsin

Kawamura, Shiho; Gerstung, Moritz; Colozo, Alejandro T.; Helenius, Jonne; Maeda, Akiko; Beerenwinkel, Niko; Park, Paul Shin-Hyun; Müller, Daniel J.

doi:10.1016/j.str.2013.01.011

Cited by 48 publications

(101 citation statements)

References 49 publications

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“…5 and 8). Such a dramatic change in lifetime induced by substrate binding is not observed for other membrane proteins-including transportersstudied thus far (24,30,32,35,61). Fast transition rates between different states are intuitively indicative of facile energy landscapes and suggest a relaxed conformational ensemble (Fig.…”

Section: Smfs Reveals 10 Structural Segments In Lacymentioning

confidence: 82%

“…2 C and D). To determine the mean contour lengths of the characteristic force peaks, all peaks from each histogram were simultaneously fitted using a Gaussian mixture model (35). The 10 characteristic force peaks recorded upon unfolding WT LacY from the elongated C-terminal end occur at mean contour lengths of 47,64,85,120,175,204,270,316,338, and 404 aa (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Linear regression was used for fitting the DFS plots, and the error propagation of the uncertainties was computed using Monte Carlo simulations (SI Appendix, Fig. S6) (35). This approach has the advantage of correctly accounting for nonlinearities and correlations among measurement errors.…”

Section: Resultsmentioning

confidence: 99%

“…Because SMFS can be used with membrane proteins embedded in native or synthetic lipid membranes under physiological conditions, the method has been used to assess interactions that change upon substrate binding, insertion of mutations, and assembly or lipid composition of the membrane (26)(27)(28)(29)(30)(31)(32)(33)(34)(35). Moreover, when operated in the dynamic mode, dynamic single-molecule force spectroscopy (DFS) localizes and quantifies the kinetic, energetic,…”

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confidence: 99%

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Substrate-induced changes in the structural properties of LacY

Serdiuk

Madej²,

Sugihara³

et al. 2014

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

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The lactose permease (LacY) of Escherichia coli, a paradigm for the major facilitator superfamily, catalyzes the coupled stoichiometric translocation of a galactopyranoside and an H + across the cytoplasmic membrane. To catalyze transport, LacY undergoes large conformational changes that allow alternating access of sugarand H + -binding sites to either side of the membrane. Despite strong evidence for an alternating access mechanism, it remains unclear how H + -and sugar-binding trigger the cascade of interactions leading to alternating conformational states. Here we used dynamic single-molecule force spectroscopy to investigate how substrate binding induces this phenomenon. Galactoside binding strongly modifies kinetic, energetic, and mechanical properties of the N-terminal 6-helix bundle of LacY, whereas the C-terminal 6-helix bundle remains largely unaffected. Within the N-terminal 6-helix bundle, the properties of helix V, which contains residues critical for sugar binding, change most radically. Particularly, secondary structures forming the N-terminal domain exhibit mechanically brittle properties in the unbound state, but highly flexible conformations in the substrate-bound state with significantly increased lifetimes and energetic stability. Thus, sugar binding tunes the properties of the N-terminal domain to initiate galactoside/H + symport. In contrast to wild-type LacY, the properties of the conformationally restricted mutant Cys154➝Gly do not change upon sugar binding. It is also observed that the single mutation of Cys154➝Gly alters intramolecular interactions so that individual transmembrane helices manifest different properties. The results support a working model of LacY in which substrate binding induces alternating conformational states and provides insight into their specific kinetic, energetic, and mechanical properties.atomic force microscopy | membrane | transport protein | membrane protein structure | membrane protein folding | membrane transport T he lactose permease of Escherichia coli (LacY) of the major facilitator superfamily (MFS) (1, 2) catalyzes the coupled stoichiometric translocation of a galactopyranoside and an H + (sugar/H + symport) (3-6). Uphill (i.e., active) symport of galactoside against a concentration gradient is achieved by transduction of free energy released from the downhill movement of H + with the electrochemical H + gradient (Δμ H + ; interior negative and/or alkaline). Conversely, because coupling between sugar and H + is obligatory, downhill galactoside transport from a high to a low sugar concentration is coupled to uphill H + transport with the generation of Δμ H +, the polarity of which depends upon the direction of the sugar concentration gradient (7-10).LacY monomers reconstituted into proteoliposomes are functional (11, 12), and X-ray crystal structures reveal 12, mostly irregular, transmembrane α-helices organized into two pseudosymmetrical 6-helix bundles surrounding a large interior hydrophilic cavity open to the cytoplasm (13-16). At the apex of the h...

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Section: Smfs Reveals 10 Structural Segments In Lacymentioning

confidence: 82%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Substrate-induced changes in the structural properties of LacY

Serdiuk

Madej²,

Sugihara³

et al. 2014

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

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“…A recent review provides details of studies that have employed the Zhurkov-Bell model to extract parameters of the unfolding energy landscape of a protein from experimental data on polyproteins [43]. In addition, since that review, 12 additional studies have used the Zhurkov-Bell model to extract information from force spectroscopy experiments [15,[44][45][46][47][48][49][50][51][52][53][54]. Given the prevalence of the Zhurkov-Bell model in force spectroscopy experiments, it is interesting to examine its application and accuracy in more detail.…”

Section: Introductionmentioning

confidence: 99%

Optimizing the calculation of energy landscape parameters from single-molecule protein unfolding experiments

et al. 2015

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Single-molecule force spectroscopy using an atomic force microscope (AFM) can be used to measure the average unfolding force of proteins in a constant velocity experiment. In combination with Monte Carlo simulations and through the application of the Zhurkov-Bell model, information about the parameters describing the underlying unfolding energy landscape of the protein can be obtained. Using this approach, we have completed protein unfolding experiments on the polyprotein (I 27) 5 over a range of pulling velocities. In agreement with previous work, we find that the observed number of protein unfolding events observed in each approach-retract cycle varies between one and five, due to the nature of the interactions between the polyprotein, the AFM tip, and the substrate, and there is an unequal unfolding probability distribution. We have developed a Monte Carlo simulation that incorporates the impact of this unequal unfolding probability distribution on the median unfolding force and the calculation of the protein unfolding energy landscape parameters. These results show that while there is a significant, unequal unfolding probability distribution, the unfolding energy landscape parameters obtained from use of the Zhurkov-Bell model are not greatly affected. This result is important because it demonstrates that the minimum acceptance criteria typically used in force extension experiments are justified and do not skew the calculation of the unfolding energy landscape parameters. We further validate this approach by determining the error in the energy landscape parameters for two extreme cases, and we provide suggestions for methods that can be employed to increase the level of accuracy in single-molecule experiments using polyproteins.

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Understanding the Rhodopsin Worldview Through Atomic Force Microscopy (AFM): Structure, Stability, and Activity Studies

Senapati

Park

2023

The Chemical Record

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Rhodopsin is a G protein‐coupled receptor (GPCR) present in the rod outer segment (ROS) of photoreceptor cells that initiates the phototransduction cascade required for scotopic vision. Due to the remarkable advancements in technological tools, the chemistry of rhodopsin has begun to unravel especially over the past few decades, but mostly at the ensemble scale. Atomic force microscopy (AFM) is a tool capable of providing critical information from a single‐molecule point of view. In this regard, to bolster our understanding of rhodopsin at the nanoscale level, AFM‐based imaging, force spectroscopy, and nano‐indentation techniques were employed on ROS disc membranes containing rhodopsin, isolated from vertebrate species both in normal and diseased states. These AFM studies on samples from native retinal tissue have provided fundamental insights into the structure and function of rhodopsin under normal and dysfunctional states. We review here the findings from these AFM studies that provide important insights on the supramolecular organization of rhodopsin within the membrane and factors that contribute to this organization, the molecular interactions stabilizing the structure of the receptor and factors that can modify those interactions, and the mechanism underlying constitutive activity in the receptor that can cause disease.

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Kinetic, Energetic, and Mechanical Differences between Dark-State Rhodopsin and Opsin

Cited by 48 publications

References 49 publications

Substrate-induced changes in the structural properties of LacY

Substrate-induced changes in the structural properties of LacY

Optimizing the calculation of energy landscape parameters from single-molecule protein unfolding experiments

Understanding the Rhodopsin Worldview Through Atomic Force Microscopy (AFM): Structure, Stability, and Activity Studies

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