Oriented proteins

Biological membranes provide the essential barrier between cells and the organelles of which cells are composed. Cellular membranes are complicated extensive biomolecular sheetlike structures, mostly fonned by lipid molecules held together by cooperative nonco-valent interactions. A membrane is not a static structure, but rather a complex dynamical two-dimensional liquid crystalline fluid mosaic of oriented proteins and lipids. A number of experimental approaches can be used to investigate and characterize biological membranes. However, the complexity of membranes is such that experimental data remain very difficult to interpret at the microscopic level. In recent years, computational studies of membranes based on detailed atomic models, as summarized in Chapter 21, have greatly increased the ability to interpret experimental data, yielding a much-improved picture of the structure and dynamics of lipid bilayers and the relationship of those properties to membrane function [21]. 

Two-dimensional protein layer orientation could be also effected by metal-ion coordination Monolayer of iminodiacetate-Cu(II) lipid was successfully employed as substrate for oriented immobilization of proteins naturally displaying histidine residues on their surface [37]. Affmity-resin-displaying Ni(II) complexes could also be successfully employed for oriented protein immobilization [38]. 

The main advantage of NMR spectroscopy is its use with proteins in solution. In consequence, rather than obtaining a single three-dimensional structure of the protein, the final result for an NMR structure is a set of more or less overlying structures which fulfill the criteria and constraints given particularly by the NOEs. Typically, flexibly oriented protein loops appear as largely diverging structures in this part of the protein. Likewise, two distinct local conformations of the protein are represented by two differentiated populations of NMR structures. Conformational dynamics are observable on different time scales. The rates of equilibration of two (or more) substructures can be calculated from analysis of the line shape of the resonances and from spin relaxation times Tj and T2, respectively. 

Other novel bicelle systems composed of a mixture of l,2-di-0-dodecyl-sn-glycero-3-phosphocholine and 3-(chloramidopropyl)dimethylammonio-2-hydroxyl-l-propane sulfonate have been designed to orient proteins at low pH values and over wide temperature ranges [25]. 

Post-Translational Lipidation of Extracellularly Oriented Proteins 537... 

Fig. 2. Schematic of the synthesis of nanostructured metals and alloys in varying grain sizes supported on hollow, highly oriented protein templates (Behrens, Dinjus, and Unger, 1999 Behrens et al., 2000).

Schneider, G., Schuchhardt, J., and Wrede, P. (1994). Artificial neural networks and simulated molecular evolution are potential tools for sequence-oriented protein design. Comput Appl Biosci 10,635-45. 

Sandoa, C. R., and Schwonek, J. P. (1993). Simulation of Solid State NMR Parameters from Oriented Proteins Progress in Algorithm Development for Total Structural Studies. Biophys. J. 65,1460-1469. 

Membranes are fuid structures. Lipid molecules diffuse rapidly in the plane of the membrane, as do proteins, unless they are anchored by specific interactions. In contrast, lipid molecules and proteins do not readily rotate across the membrane. Membranes can be regarded as two-dimensional solutions of oriented proteins and lipids. 

The wide angle x-ray diffraction pattern of undeformed corneum exhibits diffuse halos at 4.6 A and 9.8 A common to proteins (Figure 4). The lack of the 5.1-A reflection characteristic of alpha-keratin structures in undeformed comeum suggests that the protein is considerably less oriented and perhaps of a lower alpha content than wool. This is supported by the fact that the 5.1-A reflection begins to appear in samples of comeum which were hydrated and stretched to 100% or more (Figure 6) and allowed to dry in the extended state. The increased orientation of the lipid reflections in the stretched sample demonstrates further their association with the orienting protein fibrils. 

PISA wheels and dipolar waves for oriented proteins... 

As mentioned above, the determination of atomic level structure, i.e., the backbone torsion angles for an oriented protein fiber, is possible by using both solid-state NMR method described here and specifically isotope labeling. This is basically to obtain the angle information. Another structural parameter is distance between the nuclei for atomic coordinate determination. The observation of Nuclear Overhauser Enhancements (NOEs) between hydrogen atoms is a well known technique to determine the atomic coordinates of proteins in solution [14]. In the field of solid-state NMR, REDOR (rotational echo double resonance) for detection of weak heteronuclear dipole interactions, such as those due to C and N nuclei [15, 16] or R (rotational resonance) for detection of the distance between homonuclei, are typical methods for internuclear distance determination [17,18]. The REDOR technique has been applied to structure determination of a silk fibroin model compound [19]. In general, this does not require orientation of the samples in the analysis, but selective isotope labeling between specified nuclear pairs in the samples is required which frequently becomes a problem. A review of these approaches has appeared elsewhere [16]. 

The determination of polymer structure at the atomic level is possible by analyzing orientation-dependent NMR interactions such as dipole-dipole, quadrupole and chemical shielding anisotropy as mentioned above. The outline of the atomic coordinate determination for oriented protein fibers used here is described more fully in Ref. [30]. The chemical shielding anisotropy (CSA) interaction for N nucleus in an amide (peptide) plane can be interpreted with the chemical shielding tensor transformation as shown in Fig. 8.3. [31, 32]. 

The F/y terms are the i, j components of the CSA tensor expressed in the FAS reference frame. Equation (8.11) represents a family of curves that are manifested in the spectral line shape. The spectra obtained from oriented protein fiber samples aligned parallel and perpendicular to the magnetic field yield eight possible orientations of the peptide plane relative to the long axis of the fiber [31]. Note that it is necessary to consider only one set of Op, jSp angles per site in the spectral simulations since the remaining seven possible pairs for each site will result in identical lineshapes. 

The determination of the molecular frame with respect to the oriented axis is possible from the observation of the dipolar coupling constant of uniaxially oriented [1- C]—[ N] double labeled silk fiber. Namely, when the dipolar splitting is observed for the uniaxially oriented protein... 

Protein modification in solution To control and favorably orientate proteins on the chip surface for optimal analyte binding... 

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