Protein membrane-bound

The modem era of biochemistry and molecular biology has been shaped not least by the isolation and characterization of individual molecules. Recently, however, more and more polyfunctional macromolecular complexes are being discovered, including nonrandomly codistributed membrane-bound proteins [41], These are made up of several individual proteins, which can assemble spontaneously, possibly in the presence of a lipid membrane or an element of the cytoskeleton [42] which are themselves supramolecular complexes. Some of these complexes, e.g. snail haemocyanin [4o], are merely assembled from a very large number of identical subunits vimses are much larger and more elaborate and we are still some way from understanding the processes controlling the assembly of the wonderfully intricate and beautiful stmctures responsible for the iridescent colours of butterflies and moths [44]. 

The development of efficient algorithms and the sophisticated description of long-range electrostatic effects allow calculations on systems with 100 000 atoms and more, which address biochemical problems like membrane-bound protein complexes or the action of molecular machines . 

Alpha helices are sufficiently versatile to produce many very different classes of structures. In membrane-bound proteins, the regions inside the membranes are frequently a helices whose surfaces are covered by hydrophobic side chains suitable for the hydrophobic environment inside the membranes. Membrane-bound proteins are described in Chapter 12. Alpha helices are also frequently used to produce structural and motile proteins with various different properties and functions. These can be typical fibrous proteins such as keratin, which is present in skin, hair, and feathers, or parts of the cellular machinery such as fibrinogen or the muscle proteins myosin and dystrophin. These a-helical proteins will be discussed in Chapter 14. 

In this chapter we describe some examples of structures of membrane-bound proteins known to high resolution, and outline how the elucidation of these structures has contributed to understanding the specific function of these proteins, as well as some general principles for the construction of membrane-bound proteins. In Chapter 13 we describe some examples of the domain organization of receptor families and their associated proteins involved in signal transduction through the membrane. 

The reaction center is built up from four polypeptide chains, three of which are called L, M, and H because they were thought to have light, medium, and heavy molecular masses as deduced from their electrophoretic mobility on SDS-PAGE. Subsequent amino acid sequence determinations showed, however, that the H chain is in fact the smallest with 258 amino acids, followed by the L chain with 273 amino acids. The M chain is the largest polypeptide with 323 amino acids. This discrepancy between apparent relative masses and real molecular weights illustrates the uncertainty in deducing molecular masses of membrane-bound proteins from their mobility in electrophoretic gels. 

Figure 12.12 X-ray diffraction pattern from crystals of a membrane-bound protein, the bacterial photosynthetic reaction center. (Courtesy of H. Michel.)...

Figure 12.14 The three-dimensional structure of a photosynthetic reaction center of a purple bacterium was the first high-resolution structure to be obtained from a membrane-bound protein. The molecule contains four subunits L, M, H, and a cytochrome. Subunits L and M bind the photosynthetic pigments, and the cytochrome binds four heme groups. The L (yellow) and the M (red) subunits each have five transmembrane a helices A-E. The H subunit (green) has one such transmembrane helix, AH, and the cytochrome (blue) has none. Approximate membrane boundaries are shown. The photosynthetic pigments and the heme groups appear in black. (Adapted from L. Stryer, Biochemistry, 3rd ed. New York ...

Important novel information has thus been obtained for the specific biological function of those molecules, but disappointingly few general lessons have been learned that are relevant for other membrane-bound proteins with different biological functions. In that respect the situation is similar to the failure of the structure of myoglobin to provide general principles for the construction of soluble protein molecules as described in Chapter 2. 

The three-dimensional structure of the bacterial membrane protein, bac-teriorhodopsin, was the first to be obtained from electron microscopy of two-dimensional crystals. This method is now being successfully applied to several other membrane-bound proteins. 

G-proteins, trim eric membrane-bound proteins that have intrinsic GTPase activity and act as intermediaries between 7TM receptors and a host of cellular effectors see Section 2.2. 

From the human genome project it is known, that roughly 30,000 proteins exist in humans. Currently only the 3D-structures of few thousand human pr oteins or protein domains are known. Structures of membrane-bound proteins are several magnitudes rarer. Beside efforts to solve further structures like structural genomics, there is a challenge for computational approaches to predict structures and function for homologous proteins. 

SNAREs is an acronym for soluble NSF acceptor protein receptors. They are a superfamily of small and mostly membrane-bound proteins that are distinguished by the presence of a conserved stretch of 60 amino acids referred to as a SNARE motif. With few exceptions, a single transmembrane domain is located adjacent to the SNARE motif at the C-terminal end. Many SNAREs possess in addition an independently folded N-terminal domain whose structures are more diverse. 

Fig. 3 Binding and release of tropoelastin. The elastin receptor consists of a 67 kDa peripheral subunit (EBP) with two transmembrane proteins of 61 and 55 kDa. The 67 kDa protein binds tropoelastin and galactosugars through two separate sites, (a) Tropoelastin binds to the intact EBP complex, (b) Upon binding of a galactosugar, the EBP loses its affinity for both tropoelastin and the membrane-bound protein, which leads to the release of tropoelastin. Reproduced from [8] with permission from John Wiley and Sons, copyright 1998...

Until recently, the possibility that H,K-ATPase consists not only of a catalytic a subunit but also of other subunits was not examined. This was mainly due to the fact that SDS-PAGE of purified gastric H,K-ATPase preparations principally gave one protein band with an apparent molecular mass of about 100 kDa, which was reported to comprise 75% or more of the total amount of protein [6,66,67]. This mass is lower than the mass deduced from its cloned cDNA [40], but may be due to the higher electrophoretic mobility of membrane-bound proteins, as consequence of having relatively high contents of hydrophobic amino acid residues [68]. 

In bacteria, accumulation of substrates against a concentration gradient can occur through two main classes of transport systems (see [30] for a summary). The prototype of the first class of transporters is the /3-galactoside permease of Escherichia coli (see [31]). It is a relatively simple system involving only a single membrane-bound protein. It catalyzes a lactose-H symport. Other transporters... 

Imaoka, S. and Funae, Y., Ion-exchange high-performance liquid chromatography of membrane-bound protein cytochrome P-450,. Chromatogr., 375,83,1986. 

All the aforementioned protein members of the cannabinoid system are large, membrane-bound proteins therefore, it is particularly difficult to obtain direct information about their tertiary structure. Thus, at the present time, structure-based drug design is not feasible. Detailed exploration of the SAR and subsequent ligand-based design are the most appropriate means for the development of molecular probes for these proteins. 

Carotenoids are also present in animals, including humans, where they are selectively absorbed from diet (Furr and Clark 1997). Because of their hydrophobic nature, carotenoids are located either in the lipid bilayer portion of membranes or form complexes with specific proteins, usually associated with membranes. In animals and humans, dietary carotenoids are transported in blood plasma as complexes with lipoproteins (Krinsky et al. 1958, Tso 1981) and accumulate in various organs and tissues (Parker 1989, Kaplan et al. 1990, Tanumihardjo et al. 1990, Schmitz et al. 1991, Khachik et al. 1998, Hata et al. 2000). The highest concentration of carotenoids can be found in the eye retina of primates. In the retina of the human eye, where two dipolar carotenoids, lutein and zeaxan-thin, selectively accumulate from blood plasma, this concentration can reach as high as 0.1-1.0mM (Snodderly et al. 1984, Landrum et al. 1999). It has been shown that in the retina, carotenoids are associated with lipid bilayer membranes (Sommerburg et al. 1999, Rapp et al. 2000) although, some macular carotenoids may be connected to specific membrane-bound proteins (Bernstein et al. 1997, Bhosale et al. 2004). 

Inositol triphosphate (IP3)-gated channels are also associated with membrane-bound receptors for hormones and neurotransmitters. In this case, binding of a given substance to its receptor causes activation of another membrane-bound protein, phospholipase C. This enzyme promotes hydrolysis of phosphatidylinositol 4,5-diphosphate (PIP2) to IP3. The IP3 then diffuses to the sarcoplasmic reticulum and opens its calcium channels to release Ca++ ions from this intracellular storage site. 

A major limitation of diffraction techniques has been the need to obtain crystalline samples. If scientists could learn how to crystallize large molecules in a routine manner, a breakthrough would result. In the biological area, this limitation is keenly experienced for membrane-bound proteins, which are important in many biological functions. Scientists are now devising techniques and strategies to crystallize these proteins—if not in three-dimensional, then in two-dimensional lattices. 

A bacterial phosphatidylinositol specific phospholipase C (PI-PLC) had been available for many years before it was demonstrated to strip a number of membrane-bound proteins from eukaryotic cell surfaces [1], Such proteins are anchored by a PI moiety in which the 6 position of inositol is glycosidically linked to glucosamine, which in turn is bonded to a polymannan backbone (Fig. 3-10). The polysaccharide chain is joined to the carboxyl terminal of the anchored protein via amide linkage to ethanolamine phosphate. The presence of a free NH2 group in the glucosamine residue makes the structure labile to nitrous acid. Bacterial PI-PLC hydrolyzes the bond between DAG and phosphati-dylinositols, releasing the water-soluble protein polysac charide-inositol phosphate moiety. These proteins are tethered by glycosylphosphatidylinositol (GPI) anchors. 

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