Glucose-stimulated insulin secretion is a complex process that is regulated via complex signaling pathways within the cell. The dynamics of cellular signaling, and corresponding insulin release, are critical to normal regulation of serum glucose, however, detection of many cellular signals is not possible due to a dearth of sensing technologies. Recent studies have rediscovered the importance of the?
A better understanding of intracellular signaling and corresponding regulated release within both of these cell types is of paramount importance to elucidate the root causes of secretory abnormalities and the corresponding roles in the onset and progression of diabetes. The primary focus of this proposal is to develop suitable capabilities to monitor metabolic and carbohydrate-derived signals in?
We will develop, characterize and utilize a highly-stable, porous phospholipid architecture with enhanced mass transport capabilities for detection of intracellular regulators that lack intrinsic optical or electrochemical activity.
Phospholipid scaffolds are prepared with ca. This architecture will allow novel enzymatic and fluorescent reporter chemistries to be used for intracellular measurements of heretofore undetectable analytes. Onc fully-developed, porous lipid architectures may prove valuable for a host of other applications including large molecule drug delivery, etc.
This research project will lead to development of a new category of nanometer-sized chemical and biological sensors that are compatible with the intracellular environment. These sensors will enable new questions to be asked and answered in the area of signal transduction in important neuroendocrine cells and will enable new hypotheses to be tested in the role of metabolic coupling in pancreatic alpha cells and a range of other cellular systems involved in the regulation of normal human health.
Toggle navigation. For example, myelin sheath membranes, which play an electrical insulator role about neurons in animals, are enriched in lipids. Many other membranes actually contain more protein than lipid, reflecting their many roles in solute transport, catalysis, signaling, etc. The lipid compositions of the plasma membrane and organelle membranes of a rat hepatocyte are given in Fig.
In hepatocytes, the plasma membrane is enriched in cholesterol due to its close contact with lipoproteins in the blood. On the other hand, cholesterol makes up only a small percentage of mitochondrial membrane lipids.
In most cases, the functional significance of variations in membrane lipid composition remain unknown. Lastly, the protein composition of membranes varies even more widely than the lipid composition and is determined by the functional specialization of the membrane.
In this model, proteins form the ceramic tiles that float in the lipid mortar. The whole structure is held together by noncovalent interactions and the hydrophobic effect.
Due to the lack of covalent interactions between components, both lipids and proteins are able to undergo lateral diffusion in the bilayer. The acyl chains of membrane lipids are maintained in their melted states to allow lateral diffusion of components.
On the other hand, movement of lipids, and particularly proteins, from one bilayer leaflet to the other is restricted. The carbohydrate moieties of both glycolipids and glycoproteins face outside the cell, and the two sides of the bilayer are said to be asymmetrical.
We will further discuss the various features of the fluid mosaic properties of membranes, and will note some exceptions and refinements. Amphipathic Lipid Aggregates in Water The major amphipathic structural lipids of membranes-glycerophospholipids, sphingolipids, and sterols--form microscopic aggregates when placed in water.
The most common structure formed is the bilayer, which is unstable unless its edges are curved around and sealed as in vesicles liposomes. Bilayers form for the above lipids whose head groups and acyl chains have roughly the same cross-sectional areas.
Lipids such as free fatty acids and detergents such as SDS form spherical micelles since the crosssectional areas of their head groups are larger than their acyl chains. The formation of these structures is driven by the hydrophobic effect. The hydrocarbon core is about as nonpolar as decane.
Pure lipid vesicles made in the laboratory are essentially impermeable to polar solutes. Asymmetric Distribution of Phospholipids in Cell Membranes Plasma membrane lipids are asymmetrically distributed between the two leaflets of the bilayer. However, the asymmetry is not absolute as it is for membrane proteins.
The distribution of phospholipids between the inner and outer monolayers leaflets of the plasma membrane of erythrocytes is shown in Fig. The table shows that the choline-containing lipids, phosphatidylcholine and sphingomyelin, are typically found in the outer leaflet. In contrast, the amino group containing phospholipids, phosphatidylethanolamine and phosphatidylserine, along with other phospholipids are primarily found in the inner leaflet.
The transbilayer distribution of phospholipids is maintained by specific proteins see below. Distribution of Lipids in a Typical Eukaryotic Cell Each membrane in a cell has its own characteristic composition. As membrane vesicles transport components through the cell from the ER to the Golgi, then on to the plasma membrane, changes occur in vesicular lipid composition and the distribution across the membranes.
For instance, phosphatidylcholine is the major phospholipid in the lumenal leaflet of the Golgi, and vesicles moving to the trans-Golgi network.
However, in transport vesicles moving from the trans-Golgi network to the plasma membrane, phosphatidylcholine is largely replaced by sphingolipids and cholesterol, which enter the outer leaflet of the plasma membrane on fusion of the transport vesicles with the plasma membrane. In some cells, changes in membrane lipid distribution have functional consequences.
For example, phosphatidylserine must move to the outer leaflet of the platelet plasma membrane for a platelet to participate in blood coagulation. Classification of Membrane Proteins Membrane proteins are classified into three broad categories that relate to conditions needed to remove them from bilayers.
Integral membrane proteins are deeply embedded in the bilayer and have multiple hydrophobic amino acid residues in contact with bilayer lipids. They can only be removed from the membrane by reagents, such as detergents, that disrupt hydrophobic interactions between the protein and membrane lipids and coat the hydrophobic domain with a micellar like phase. Peripheral membrane proteins are associated with the membrane through electrostatic and hydrogen bonding interactions to other membrane proteins or the polar head groups of phospholipids.
They can readily be removed from the membrane by changing the pH or ionic strength of the solution. Finally, amphitrophic protein attachment to membranes is regulated by a biological modification such as attachment of a lipid anchor or phosphorylation. Like peripheral membrane proteins, they are not embedded in the bilayer. Membrane Protein Topology I The term membrane protein topology refers to the localization of protein sequences and domains with respect to the plane of the lipid bilayer.
All copies of a given protein adopt the same topology in the membrane, and are oriented asymmetrically with respect to the bilayer plane. The topology of the erythrocyte plasma membrane protein, glycophorin, is shown in Fig.
The N-terminus of glycophorin resides outside the membrane, while the C-terminus is located in the cytoplasm. The attachment points for N- and O-linked oligosaccharide chains are located in the polar region located outside the membrane. Glycosylated domains are invariably found outside the membrane in membrane glycoproteins. Types I and II have a single transmembrane helix, where the C-terminal end of the protein is located outside or inside, respectively. Note that glycophorin is a Type II integral membrane protein.
Type III proteins have multiple transmembrane segments, while Type IV proteins are assemblies of multiple different polypeptide chains usually oriented to form a channel in the bilayer. Type V membrane proteins are held to the bilayer primarily by covalently linked lipids. Lastly, Type VI membrane proteins have both transmembrane segments and lipid anchors. The structures of very few integral membrane proteins have been solved by X-ray crystallography.
Usually structures are determined by treatment with proteases or chemical reagents that are membrane impermeable and modify amino acids on only one side of the bilayer. V IV Structure of Bacteriorhodopsin The integral membrane protein called bacteriorhodopsin is one of few membrane proteins whose structure has been determined at atomic detail by X-ray crystallography. Bacteriorhodopsin is a light-driven proton pump that is located in the purple membranes of the photosynthetic bacterium, Halobacterium salinarum.
Conformational changes in retinal caused by light absorption drive conformational changes in apobacteriorhodopsin which cause protons to be pumped across the membrane.
This generates a proton gradient across the membrane which is exploited for energy production. The seven transmembrane segments are tilted slightly with respect to the plane of the membrane. The seven transmembrane segments are clustered together and are surrounded by membrane lipids. Some membrane lipids actually occupy spaces between the segments. Lipid Annuli Surrounding Membrane Proteins Included in the X-ray crystal structures of integral membrane proteins are tightly bound membrane lipids surrounding the proteins as a bilayer shell or annulus.
It is presumed that these lipids also bind tightly to the proteins in membranes. In Fig. In the figure, aquaporin protein is colored dark blue.
Tightly bound lipids have their head groups colored in light blue and their fatty acyl chains in yellow.
The polar head groups of the lipids contact polar amino acids in aquaporin, whereas the fatty acyl chains contact hydrophobic residues. It is not uncommon for a membrane protein to require the presence of certain lipids in the annulus surrounding it. Membrane Protein Topology Prediction I The locations of transmembrane segments in a membrane protein can be predicted from computer-based analysis of its amino acid sequence.
In essence, what is done is that an algorithm is used to scan the amino acid sequence looking for stretches of residues on the order of 20 amino acids long that are sufficiently nonpolar to be embedded in a lipid bilayer.
To achieve this end, amino acids are assigned a hydropathy index for transfer from water to a hydrocarbon phase such as listed in Table Then the membrane protein sequence is scanned and the average hydropathy index is calculated for a window of 7 to 20 residues, depending on the program. The average hydropathy is plotted on the y-axis against the middle residue number in the window.
Plots like those shown in Fig. In this figure it can be seen that the sequence of glycophorin yields one predicted transmembrane segment, whereas the sequence of bacteriorhodopsin yields 6 or 7 segments. First, as the crystal structures of the five membrane proteins in Fig. These residues serve as membrane interface anchors because they are simultaneously able to interact with the lipid and aqueous phases on either side of the bilayer surface.
All backbone hydrogen bonds occur in the interior of the helix, and the R groups radiate out from the helix axis; if composed of nonpolar residues, the helix is ideally designed for interaction with membrane fatty acyl chains. And in most cases every other residue in the strands is a nonpolar one whose R group points out toward membrane fatty acyl chains. Lipid-linked Membrane Proteins Some membrane proteins contain one or more covalently attached lipids that anchor them to membranes Fig.
Lipid-linked membrane proteins can be attached to the membrane via palmitoyl groups covalently bound to an internal Cys or Ser residue, an N-myristoyl group on an N-terminal Gly residue, a farnesyl or geranylgeranyl group on a C-terminal Cys residue, or a GPI glycosylated phosphatidylinositol linkage. The first three types of lipid-linked proteins reside in the cytoplasm. GPI-linked membrane proteins project outside the cell.
N-myristoylated proteins commonly also contain a hydrophobic transmembrane segment. Cysteine-palmitoyl-linked proteins are weakly attached to the membrane and membrane binding often is reversible. In intestinal epithelial cells, GPI-linked proteins are exclusively targeted to the apical lumenal instead of basal bloodstream membranes of the cells. Phase Transitions in Membrane Lipids Membrane lipids have characteristic melting points that are determined largely by the composition of their fatty acids.
Saturated fatty acids pack better next to one another in bilayers than do unsaturated and polyunsaturated fatty acids. Therefore, bilayer lipids rich in saturated fatty acids are less fluid than lipids rich in unsaturated fatty acids at a given temperature. For any membrane lipid composition, lowering the temperature significantly will create what is termed the liquid-ordered state, wherein fatty acyl chains are extended and thermal motions are greatly restricted Fig.
At significantly higher temperatures, carboncarbon single bonds in fatty acyl chains begin to rotate, chains become less extended, and thermal motions become considerable liquid-disordered state. Lipids become free to rotate and diffuse laterally in the bilayer. All cells regulate the degree of fluidity of their membrane lipids see Fig. Animals regulate fluidity by adjusting the levels of unsaturated fatty acids and cholesterol.
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