The interaction of water molecules with purple membrane suspension using ²H double‐quantum filter, ¹H and ²H diffusion nuclear magnetic resonance

Abstract

Bacteriorhodopsin is a membrane protein of the purple membrane (PM) of Halobacterium salinarum, which is isolated as sheets of highly organized two-dimensional hexagonal microcrystals and for which water molecules play a crucial role that affects its function as a proton pump. In this paper we used single- and double-quantum (2)H NMR as well as (1)H and (2)H diffusion NMR to characterize the interaction of water molecules with the PM in D(2)O suspensions. We found that, under the influence of a strong magnetic field on a concentrated PM sample (0.61 mM), the PM sheets affect the entire water population and a residual quadrupolar splitting (upsilon(q) approximately 5.5 Hz, 298 K, at 11.7 T) is observed for the D(2)O molecules. We found that the residual quadrupolar coupling, the creation time in which a maximal DQF signal was obtained (tau(max)), and the relative intensity of the (2)H DQF spectrum of the water molecules in the PM samples (referred to herein as NMR order parameters) are very sensitive to temperature, dilution, and chemical modifications of the PM. In concentrated PM samples in D(2)O, these NMR parameters seem to reflect the relative organization of the PM. Interestingly, we have observed that some of these parameters are sensitive to the efficiency of the trimer packing, as concluded from the apo-membrane behavior. The data for dionized blue membrane, partially delipidated sample, and detergent-treated PM show that these D(2)O NMR order parameters, which are magnetic field dependent, are sensitive to the structural integrity of the PM. In addition, we revealed that heating the PM sample inside or outside the NMR magnet has, after cooling, a different effect on the NMR characteristics of the water molecules in the concentrated PM suspensions. The difference in the D(2)O NMR order parameters for the PM samples, which were heated and cooled in the presence and in the absence of a strong magnetic field, corroborates the conclusions that the above D(2)O order parameters are indirect reflections of both microscopic and macroscopic order of the PM samples. In addition, (1)H NMR diffusion measurements showed that at least three distinct water populations could be identified, based on their diffusion coefficients. These water populations seem to correlate with different water populations previously reported for the PM system.

Simulation studies of instrumental artifacts on spin I=1 double quantum filtered NMR spectroscopy

“…The method has been implemented with great success in a wide range of systems of biological relevance. For example, the technique has been used to study motional anisotropy of water in blood vessels [2], the dynamics of water in tendon [3], the interaction of water within a purple membrane suspension [4], to detect anisotropy in cartilage [5] [6] and to discriminate between various compartments in sciatic nerve [7]. More recently the experimental scheme has been applied to study spinal disc degeneration [8].…”

Effects of Instrumental Artifacts on Double-Quantum-Filtered NMR Buildup Curves for Spin $$I=1$$ I = 1

“…The experimental method has been successfully applied in a wide range of chemical and biological systems. For instance, this technique has been employed to study the anisotropy and the fiber orientation in cartilage [2,3], to discriminate between different compartments in sciatic nerve [4], to probe the water anisotropic motion in blood vessel walls [5], the water dynamics in tendon and in elastin [6,7] and the interaction of water with purple membrane suspension [8], as well as to investigate the spinal disc degeneration [9]. In addition, a review of the applications of the DQF NMR scheme on biological tissues is shown in Ref.…”

Insitu X-ray reflectivity studies of molecular and molecular-cluster intercalation within purple membrane films

“…4,5 In this process, water provides the main source of proton donation to bR, and so the behaviour and interaction of water molecules near the surface of the PM sheets is of particular mechanistic interest. [6][7][8][9] In many technical applications, PM sheets are stacked by controlled sedimentation into mesostructured thin lms that require some level of hydration to retain their proton-pumping functionality. 10,11 Hybrid lms formed by intercalating a guest material with the PM sheets are more structurally robust than pure PM lms, 12 but intercalation can impact on protein functionality due to inhibition of water transport through the lm.…”