Membrane proteins fluctuations

Since Ca is transferred from one side of the membrane to the other side in association with the Ca -ATPase, thermal fluctuation of critical regions of the Ca -ATPase influenced in specific ways through the phosphorylation of the enzyme by ATP may play a role in Ca translocation. Similar ideas have been proposed some time ago by Huxley [419] in relationship to crossbridge movements during muscle contraction and by Welch and others on the role of protein fluctuations in enzyme action [420-430]. 

Indeed, hydrophilic N- or C-terminal ends and loop domains of these membrane proteins exposed to aqueous phases are able to undergo rapid or intermediate motional fluctuations, respectively, as shown in the 3D pictures of transmembrane (TM) moieties of bacteriorhodopsin (bR) as a typical membrane protein in the purple membrane (PM) of Halobacterium salinarum.176 178 Structural information about protein surfaces, including the interhelical loops and N- and C-terminal ends, is completely missing from X-ray data. It is also conceivable that such pictures should be further modified, when membrane proteins in biologically active states are not always present as oligomers such as dimer or trimer as in 2D or 3D crystals but as monomers in lipid bilayers. 

TABLE 2 Estimated fluctuation changes in the transmembrane and cytoplasmic a-helices for a variety of membrane proteins and their complexes (Hz)°... 

We have emphasized here that the dynamic aspects of NMR studies are crucially important for structurally or dynamically heterogeneous systems such as synthetic or natural hydrogels, protein fibrils and membrane proteins. This is in order to characterize their unique chemical, physical and biological properties in terms of a variety of fluctuation frequencies, including high (> 108 Hz) or intermediate (104-105 Hz) frequency fluctuations. It turns out that the presence of the high-frequency motions, which are readily evaluated by comparative CPMAS and DDMAS studies, is... 

It is emphasized that revealing the dynamics as well as the structure (or conformation) based on several types of spin-relaxation times is undoubtedly a unique and indispensable means, only available from NMR techniques at ambient temperature of physiological significance. Usually, the structure data themselves are available also from X-ray diffraction studies in a more refined manner. Indeed, better structural data can be obtained at lower temperature by preventing the unnecessary molecular fluctuations, which are major subjects in this chapter, since structural data can be seriously deteriorated for domains where dynamics are predominant even in the 2D or 3D crystalline state or proteoliposome at ambient temperature. It should be also taken into account that the solubilization of membrane proteins in detergents is an alternative means to study structure in solution NMR. However, it is not always able faithfully to mimick the biomembrane environment, because the interface structure is not always the same between the bilayer and detergent system. This typically occurs in the case of PLC-81(1-140) described in Section 4.2.4 and other types of peptide systems. 

The motion of the R1 nitroxide in a protein has contributions from the overall tumbling of the protein, the internal motions of the side chain, and fluctuations in the backbone structure. For membrane proteins such as rhodopsin, the correlation time for molecular tumbling is slow on the EPR time scale defined above and can be ignored. The internal motion of the R1 side chain is due to torsional oscillations about the bonds that connect the nitroxide to the backbone, and the correlation times for these motions lie in the nanosecond regime where the EPR spectra are highly sensitive to changes in rate. 

The existence of microdomains within a membrane creates lateral heterogeneity that has consequences in terms of local concentrations of membrane-associated compounds, including membrane-active drugs, and the cooperativity between membrane proteins. It also causes fluctuations in the physicochemical properties of bilayers, including chemical interactions in the interfacial head group regions. These fluctuations can have profound effects on protein function and drug availability. 

At the moment, very little is known about the coupling of collective density fluctuations in proteins and their hydration water (and lipids, in the case of membrane proteins). New instruments recently developed for coherent neutron scattering, combined with selective sample deuteration, may fill this gap soon, and MD simulations are expected to be helpful in the interpretation of the data [60,92]. 

As an alternative means, it is a natural consequence to expect that high-resolution sohd-state NMR could be conveniently utilized to reveal the 3D structure and dynamics of a variety of membrane proteins, because the expected NMR line widths available from sohd-state NMR are not any more influenced by motional fluctuation of proteins under consideration as a whole as encountered in solution NMR. For instance, an attempt was made to determine 3D structure of uniformly C-labeled a-spectrin SH3 domain as a globular protein, based on distance constraints estimated from proton-driven spin-diffusion (PDSD) measure-... 

It should be expected, however, that C NMR signals of uniformly or densely Relabeled, fully hydrated membrane proteins could be substantially broadened or sup-pressed at ambient temperature, because their backbone or side-chain carbons of certain residues could undergo local fluctuation motions with either correlation times of the order of less than 10 or 10 -10 Rs that vary depending upon the portions under consideration, especially for flexible residues at the N- and C-terminal residues or loops, respectively, as encountered for bR as described below- Therefore, the above-mentioned approach is not always useful for fully hydrated membrane proteins that could undergo a variety of fluctuation motions. Instead, 3D structural data for transmembrane portions were successfully obtained from N NMR data of uniformly (but not densely) N-labeled proteins in either a mechanically or a magnetically aligned system, as far as such samples were prepared at relatively lower humidity. Still, no structural information is available from residues located at unoriented or flexible N- or C-terminal residues as well as at interhelical loops. 

In the case of [l- C]Val-bR, which is sensitive to fluctuation motions of 10 Hz, this condition is more stringent than that of [3- C]Ala-bR, which is sensitive to fluctuation motions of 10 Hz. A NMR spectral pattern characteristic of 2D lattice is achieved at 0°C as viewed from the [l- C]Val-labeled bR in the DPPC bilayer (Fig. 30C), while this feature is achieved from the [3- C]Ala-labeled bR at 20°C (Fig. 30B). Therefore, a suitable choice of C-labeled amino acid, either [3- C]Ala or [l- C]Val as well as the manner of protein aggregation is very important for the study of the conformation and dynamics of membrane proteins by the site-directed NMR approach, as summarized in Tables 7 and 8. 

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