Vectorially oriented membrane protein monolayers: profile structures via x-ray interferometry/holography

Chupa, Janine A.; McCauley, John P.; Strongin, Robert M.; Smith, Alexander B.; Blasie, J. Kent; Peticolas, L. J.; Bean, J. C.

doi:10.1016/s0006-3495(94)80486-2

Cited by 24 publications

(34 citation statements)

References 33 publications

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“…The SA approach was originally developed to vectorially orient detergent-solubilized integral membrane proteins at the solid-gas or solid-liquid interface [18,19], and has been extended more recently as described in Sec. II, gainfully employing the hexa-histidyl tag appended to the C terminus (or N terminus) of membrane proteins prepared by expression [22].…”

Section: Resultsmentioning

confidence: 99%

“…X-ray and neutron reflectivity, enhanced by interferometry [15–17], can be utilized to characterize the profile structures of the chemisorbed bio-organic overlayers formed on the surface of inorganic multilayer substrates at each stage of their fabrication by either the DA or SA approach [18,19]. The “profile structure” is a projection of the overlayer’s three-dimensional (3D) structure parallel to the plane of the membrane onto the normal to that plane, thereby averaging over the in-plane structure of the overlayer.…”

Section: Introductionmentioning

confidence: 99%

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Profile structures of the voltage-sensor domain and the voltage-gated K+-channel vectorially oriented in a single phospholipid bilayer membrane at the solid-vapor and solid-liquid interfaces determined by x-ray interferometry

et al. 2011

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One subunit of the prokaryotic voltage-gated potassium ion channel from Aeropyrum pernix (KvAP) is comprised of six transmembrane α helices, of which S1–S4 form the voltage-sensor domain (VSD) and S5 and S6 contribute to the pore domain (PD) of the functional homotetramer. However, the mechanism of electromechanical coupling interconverting the closed-to-open (i.e., nonconducting-to-K+-conducting) states remains undetermined. Here, we have vectorially oriented the detergent (OG)-solubilized VSD in single monolayers by two independent approaches, namely “directed-assembly” and “self-assembly,” to achieve a high in-plane density. Both utilize Ni coordination chemistry to tether the protein to an alkylated inorganic surface via its C-terminal His6 tag. Subsequently, the detergent is replaced by phospholipid (POPC) via exchange, intended to reconstitute a phospholipid bilayer environment for the protein. X-ray interferometry, in which interference with a multilayer reference structure is used to both enhance and phase the specular x-ray reflectivity from the tethered single membrane, was used to determine directly the electron density profile structures of the VSD protein solvated by detergent versus phospholipid, and with either a moist He (moderate hydration) or bulk aqueous buffer (high hydration) environment to preserve a native structure conformation. Difference electron density profiles, with respect to the multilayer substrate itself, for the VSD-OG monolayer and VSD-POPC membranes at both the solid-vapor and solid-liquid interfaces, reveal the profile structures of the VSD protein dominating these profiles and further indicate a successful reconstitution of a lipid bilayer environment. The self-assembly approach was similarly extended to the intact full-length KvAP channel for comparison. The spatial extent and asymmetry in the profile structures of both proteins confirm their unidirectional vectorial orientation within the reconstituted membrane and indicate retention of the protein’s folded three-dimensional tertiary structure upon completion of membrane bilayer reconstitution. Moreover, the resulting high in-plane density of vectorially oriented protein within a fully hydrated single phospholipid bilayer membrane at the solid-liquid interface will enable investigation of their conformational states as a function of the transmembrane electric potential.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Profile structures of the voltage-sensor domain and the voltage-gated K+-channel vectorially oriented in a single phospholipid bilayer membrane at the solid-vapor and solid-liquid interfaces determined by x-ray interferometry

et al. 2011

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“…Cyt c plays an important role in respiration (45); it has potential applications in bioelectronics (46), and was recently shown to play a role in protein-protein signaling and apoptosis (47). It is the experimental standard for studies of biological electron transfer (24,45) and has served as a model for studies of protein folding and dynamics.…”

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confidence: 99%

Gaussian fluctuations and linear response in an electron transfer protein

Simonson

2002

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

115

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In response to charge separation or transfer, polar liquids respond in a simple linear fashion. A similar linear response for proteins might be expected from the central limit theorem and is postulated in widely used theories of protein electrostatics, including the Marcus electron transfer theory and dielectric continuum theories. Although these theories are supported by a variety of experimental data, the exact validity of a linear protein dielectric response has been difficult to determine. Molecular dynamics simulations are presented that establish a linear dielectric response of both protein and surrounding solvent over the course of a biologically relevant electron transfer reaction: oxido-reduction of yeast cytochrome c in solution. Using an umbrella-sampling free energy approach with long simulations, an accurate treatment of long-range electrostatics and both classical and quantum models of the heme, good agreement is obtained with experiment for the redox potential relative to a heme-octapeptide complex. We obtain a reorganization free energy that is only half that for heme-octapeptide and is reproduced with a dielectric continuum model where the heme vicinity has a dielectric constant of only 1.1. This value implies that the contribution of protein reorganization to the electron transfer free energy barrier is reduced almost to the theoretical limit (a dielectric of one), and that the fluctuations of the electrostatic potential on the heme have a simple harmonic form, in accord with Marcus theory, even though the fluctuations of many individual protein groups (especially at the protein surface) are anharmonic. Chemical events in proteins often involve charge separation or transfer: enzyme reactions, photoexcitation of bound chromophores, proton binding and release, and electron transfer (1, 2). In response to such an event, the protein and solvent undergo dielectric relaxation, or reorganization, in the form of electronic polarization and displacement of atomic groups. Thus, enzyme reaction rates and driving forces are sensitive to the dielectric properties of the active site (3). An important goal in biophysics is to find models that can describe the dielectric properties of proteins (3-7). In the case of small molecules in aqueous solution, simple behavior is observed: the reorganization of the solvent is approximately a linear function of the solute charge rearrangement (8)(9)(10)(11)(12). This is consistent with simple models, like the dielectric continuum model (4, 13), or a model where the solvent fluctuations are assumed to be gaussian (14-16).Proteins are much more complex. They are heterogeneous polymers, and fluctuations around their folded structure are governed by an anharmonic, rugged energy surface (17, 18). The average polarity and flexibility of a folded protein are much lower than those of water, so the dielectric properties vary strongly across the protein-solvent interface. Recent experimental studies have used spectroscopic probes (19-23) whose optical absorption and emission are s...

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“…However, neither approach is as powerful as that available for phasing the specular x-ray or neutron reflectivity from thin films on solid substrates. In such cases, the solid substrate can be tailored synthetically to provide a reference structure for interferometric phasing, whether or not the distortedwave Born approximation is satisfied, [15][16][17]21 and the solutions are shown to be unique. Unfortunately, the presence of the solid substrate may perturb the system of interest in which case the box refinement and to a lesser extent the logarithmic dispersion relation approaches are superior to the slab-model refinement approach in that the phase problem is solved.…”

Section: Discussionmentioning

confidence: 99%

Solution to the phase problem for specular x-ray or neutron reflectivity from thin films on liquid surfaces

2003

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The phase problem for specular x-ray and neutron reflectivity from liquid surfaces and thin films on liquid surfaces can be solved in the distorted-wave Born approximation. The gradient of the scattering-length density ͑SLD͒ profile normal to the plane of the surface is bounded in these cases. This provides a powerful constraint allowing the phase problem to be solved with no a priori assumptions via an iterative Fourier refinement procedure applied to the Fresnel-normalized reflectivity. The critical boundary condition can be determined experimentally from the autocorrelation of the gradient profile obtained via an inverse Fourier transform of the Fresnel-normalized reflectivity without phase information. The phase solution and the resulting gradient SLD profile can be shown to be unique, and therefore unambiguously determined, when all of phase space is systematically explored for particular cases, especially for thin films on liquid surfaces. This gradient SLD profile can then be integrated either numerically, or better, analytically to provide the scattering-length density profile itself on an absolute scale.

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Vectorially oriented membrane protein monolayers: profile structures via x-ray interferometry/holography

Cited by 24 publications

References 33 publications

Profile structures of the voltage-sensor domain and the voltage-gated K+-channel vectorially oriented in a single phospholipid bilayer membrane at the solid-vapor and solid-liquid interfaces determined by x-ray interferometry

Profile structures of the voltage-sensor domain and the voltage-gated K+-channel vectorially oriented in a single phospholipid bilayer membrane at the solid-vapor and solid-liquid interfaces determined by x-ray interferometry

Gaussian fluctuations and linear response in an electron transfer protein

Solution to the phase problem for specular x-ray or neutron reflectivity from thin films on liquid surfaces

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