Membrane-bound

A typical force curve showing the specific avidin-biotin interaction is depicted in figure Bl.20.10. The SFA revealed the strong influence of hydration forces and membrane undulation forces on the specific binding of proteins to membrane-bound receptors [81]. 

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]. 

Vos M H, Jones M R, Hunter C N, Breton J, Lambry J C and Martin J L 1996 Femtosecond spectroscopy and vibrational coherence of membrane-bound RCs of Rhodobacfe/ sp/raero/des genetically modified at positions M210 and LI 81 The Reaction Center of Photosynthetic Bacteria—Structure and Dynamics ed M E Michel-Beyerle (Berlin Springer) pp 271-80... 

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 . 

Rec tor. Receptors are membrane-bound or soluble proteins or protein complexes exerting a physiological effect after binding of an agonist. 

Enzymatic Conversion of Cholesterol. A decrease of cholesterol in meat products in the future may be possible through the conversion of cholesterol [57-88-5] to coprosterol [560-68-9] which is not absorbed readily in the intestine. Cholesterol reductase can be isolated from alfalfa leaves and cucumber leaves (53). Treatment of meat animals might involve an injection of this ensyme immediately prior to slaughter, allowing for the conversion of a portion of the membrane-bound cholesterol into coprostanol. 

Active Transport. Maintenance of the appropriate concentrations of K" and Na" in the intra- and extracellular fluids involves active transport, ie, a process requiring energy (53). Sodium ion in the extracellular fluid (0.136—0.145 AfNa" ) diffuses passively and continuously into the intracellular fluid (<0.01 M Na" ) and must be removed. This sodium ion is pumped from the intracellular to the extracellular fluid, while K" is pumped from the extracellular (ca 0.004 M K" ) to the intracellular fluid (ca 0.14 M K" ) (53—55). The energy for these processes is provided by hydrolysis of adenosine triphosphate (ATP) and requires the enzyme Na" -K" ATPase, a membrane-bound enzyme which is widely distributed in the body. In some cells, eg, brain and kidney, 60—70 wt % of the ATP is used to maintain the required Na" -K" distribution. 

The ANP leceptoi exists in two forms, ANP and ANPg, both of which have been cloned. These membrane-bound guanylate cyclases have a single transmembrane domain, an intracellular protein kinase-like domain, and a catalytic cyclase domain, activation of which results in the accumulation of cychc guanosine monophosphate (cGMP). A third receptor subtype (ANP ) has been identified that does not have intrinsic guanylate cyclase activity and may play a role in the clearance of ANP. 

Plasma levels of 3—5 p.g/mL are obtained two hours after adraiinistration of 200 mg ketoconazole. No accumulation in the bloodstream was noted after a 30-wk treatment with this dose. The half-life is approximately eight hours. When ketoconazole is taken with meals, higher plasma levels are obtained. Distribution studies using radioactive ketoconazole in rats show radioactivity mainly in the Hver and the connective tissue. Radioactivity is also present in the subcutaneous tissue and the sebaceous glands. After one dose of 200 mg in humans, ketoconazole is found in urine, saUva, sebum, and cenimen. Like miconazole, the mode of action is based on inhibition of the cytochrome P-450 dependent biosynthesis of ergosterol. This results in disturbed membrane permeabiUty and membrane-bound enzymes (8,10,23,25). 

Enzyme Immunosensors. Enzyme immunosensors are enzyme immunoassays coupled with electrochemical sensors. These sensors (qv) require multiple steps for analyte determination, and either sandwich assays or competitive binding assays maybe used. Both of these assays use antibodies for the analyte of interest attached to a membrane on the surface of an electrochemical sensor. In the sandwich assay type, the membrane-bound antibody binds the sample antigen, which in turn binds another antibody that is enzyme-labeled. This immunosensor is then placed in a solution containing the substrate for the labeling enzyme and the rate of product formation is measured electrochemically. The rate of the reaction is proportional to the amount of bound enzyme and thus to the amount of the analyte antigen. The sandwich assay can be used only with antigens capable of binding two different antibodies simultaneously (53). 

Alpha helices that cross membranes are in a hydrophobic environment. Therefore, most of their side chains are hydrophobic. Long regions of hydrophobic residues in the amino acid sequence of a protein that is membrane-bound can therefore be predicted with a high degree of confidence to be transmembrane helices, as will be discussed in Chapter 12. 

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. 

Figure 12.1 Four different ways in which protein molecules may be bound to a membrane. Membrane-bound regions are green and regions outside the membrane are red. Alpha-helices are drawn as cylinders and P strands as arrows. From left to right are (a) a protein whose polypeptide chain traverses the membrane once as an a helix, (b) a protein that forms several transmembrane a helices connected by hydrophilic loop regions,...

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. 

Given the difficulty of obtaining three-dimensional crystals of membrane proteins, it is not surprising that the electron microscope technique is now widely used to study large membrane-bound complexes such as the acetylcholine receptor, rhodopsin, ion pumps, gap junctions, water channels and light-harvesting complexes, which crystallize in two dimensions. 

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 ...

The functional reaction center contains two quinone molecules. One of these, Qb (Figure 12.15), is loosely bound and can be lost during purification. The reason for the difference in the strength of binding between Qa and Qb is unknown, but as we will see later, it probably reflects a functional asymmetry in the molecule as a whole. Qa is positioned between the Fe atom and one of the pheophytin molecules (Figure 12.15). The polar-head group is outside the membrane, bound to a loop region, whereas the hydrophobic tail is... 

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 are molecular amplifiers for a large number of seven-trans-membrane helix receptors that regulate responses like vision, smell and stress response. They are heterotrimeric molecules, Gap, that dissociate into membrane-bound Ga and Gpy signal transmitters upon activation of the receptor. 

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