Deuterium nuclear magnetic resonance spectroscopy and differential scanning calorimetry are used to map the phase boundaries of mixtures of cholesterol and chain-perdeuteriated 1,2-dipalmitoyl-sn- glycero-3-phosphocholine at concentrations from 0 to 25 mol % cholesterol. Three distinct phases can be identified: the La or liquid-crystalline phase, the gel phase, and a high cholesterol concentration phase, which we call the 0 phase. The liquid-crystalline phase is characterized by highly flexible phospholipid chains with rapid axially symmetric reorientation; the gel phase has much more rigid lipid chains, and the motions are no longer axially symmetric on the 2H NMR time scale; the 0 phase is characterized by highly ordered (rigid) chains and rapid axially symmetric reorientation. In addition, we identify three regions of two-phase coexistence. The first of these is a narrow La/gel-phase coexistence region lying between 0 and about 6 mol % cholesterol at temperatures just below the chain-melting transition of the pure phospho- lipid/water dispersions, at 37.75 OC. The dramatic changes in the *H NMR line shape which occur on passing through the phase transition are used to map out the boundaries of this narrow two-phase region. The boundaries of the second two-phase region are determined by 2H NMR difference spectroscopy, one boundary lying near 7.5 mol 5% cholesterol and running from 37 down to at least 30 OC; the other boundary lies near 22 mol 5% cholesterol and covers the same temperature range. Within this region, the gel and /3 phases coexist. As the temperature is lowered below about 30 "C, the phospholipid motions reach the intermediate time scale regime of 2H NMR so that spectral subtractions become difficult and unreliable. The third two-phase region lies above 37 OC, beginning at a eutectic point somewhere between 7.5 and 10 mol % cholesterol and ending at about 20 mol %. In this region, the La and /3 phases are in equilibrium. The boundaries for this region are inferred from differential scanning calorimetry traces, for the boundary between the La- and the two-phase region, and from a dramatic sharpening of the NMR peaks on crossing the boundary between the two-phase region and the &phase region. In this region, the technique of difference spectroscopy fails, presumably because the diffusion rate in both the La- and P-phase domains is so rapid that phospholipid molecules exchange rapidly between domains on the experimental time scale.

Phase equilibria of cholesterol/dipalmitoylphosphatidylcholine mixtures: 2H nuclear magnetic resonance and differential scanning calorimetry.

Fluorescence spectroscopy, one of the most informative and sensitive analytical techniques, has played and continues to play key roles in modern research. Indeed, unraveling the inner workings of biomolecules, cells and organisms relied on the development of fluorescence-based tools. As many of the players in these sophisticated interactions and exceedingly complex systems are not inherently emissive, researchers have relied on synthesizing fluorescent analogs of the building blocks found in biological macromolecules. These are the constituents of the cell surface and cell membrane, as well as proteins and nucleic acids. This review article is dedicated to emissive analogs of these relatively small molecules.

For organizational purposes, we have arbitrarily selected to approach these diverse families of biomolecules by imagining “a journey into the center of the cell”. Approaching the exterior of a cell, one first encounters oligosaccharides that decorate the cell surface and are involved in cell recognition and signaling. Next, we arrive at the cell membrane itself. This semi-permeable envelope sets the cell boundaries and regulates its traffic. Several types of building blocks assemble this membrane, most notably among them are the phospholipids. Upon entering the cell, the cytosol reveals a plethora of small and large molecules, including proteins, as well as soluble RNA molecules and RNA-rich ribosomes. Within the cytosol of eukaryotes and prokaryotes lies the nucleus or nucleoid, respectively. This membrane-enclosed control center contains most of the cells’ genetic material. DNA, the cellular blueprint, is permanently found in the nucleus, which also hosts diverse RNA molecules. Accordingly, we first discuss emissive carbohydrate derivatives. We then present fluorescent membrane constituents, followed by emissive amino acids. Our journey ends by focusing on emissive analogs of nucleosides and nucleotides, the building blocks of nucleic acids.

The common biomolecular building blocks, excluding a few amino acids, lack appreciably useful fluorescence properties. This implies that structural modifications are required to impart such photophysical features. Ideally, a designer probe should closely resemble its natural counterpart in size and shape without the loss of the original function (a feature we refer to as “isomorphicity”). This presents a fundamental predicament, as any modification attempting to alter the electronic nature of a molecule, typically by including aromatic residues or extending conjugation, will also alter its steric bulk and therefore the interactions with its surroundings.

Clearly not all biomolecular building blocks can or need to accommodate strict isomorphic design criteria. The heterocycles found in nucleosides already provide a platform that facilitates the extension of π-conjugation, which is also true for some aromatic amino acids. In contrast, employing fluorescence spectroscopy to membrane research requires very creative probe designs. Saccharides can be viewed as the most restrictive in this context, as no chemical modification is conceivable without a major structural disruption and likely loss of function. Such aliphatic biomolecules accommodate labeling only, where an established fluorophore is covalently conjugated to provide an emissive derivative. We therefore reserve the term probe to molecular designs that are expected to furnish useful modified biomolecules capable of reliable reporting. Understandably, fluorescent probes must meet the most stringent isomorphic design principles to ensure a biologically meaningful read-out. The isomorphic design principle is therefore a central theme of this review.

This article focuses on designing fluorescent probes for the four major families of macromolecular building blocks discussed above. Although not necessarily in chronological order, it spans roughly four decades of probe design with emphasis, when justified, on recent contributions. As the reader may imagine, this topic encapsulates a vast research field and cannot be comprehensively reviewed within the space limitation of Chemical Reviews. Nevertheless, we have attempted to summarize the most important and general contributions discussing fluorescent probes that were designed to shed light on biological processes and refer the reader to other resources.1 Although a few examples have found their way into the text, we do not generally address here the development of small molecule fluorophores and sensors that are not part of biomolecular assemblies. We open this article with a brief overview of the key features of fluorescence spectroscopy, where essential theoretical, experimental, and practical elements are discussed.

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Fluorescent Analogs of Biomolecular Building Blocks: Design, Properties, and Applications

Anesthetics almost always disorder or "fluidize" membranes, i.e. the drugs increase the mobility of spin labels and reduce order parameters. This effect is universal at high concentrations above the clinical range, but in some kinds of membranes low concentrations of drugs have an ordering effect. Drugs that carry charges, including many local anesthetics, often stiffen membranes, as do long-chain alcohols or fatty acids that mimic natural membrane components. The potencies of short-chain alcohols correlate well with lipid solubility, but a cutoff is reached at 10-12 carbons, where pharmacological actions become weak or absent despite a progressive increase in lipid solubility. The cutoff is partly explained by the ordering action of the long chains and partly by the difficulty of administering such water-insoluble drugs in vivo. The idea of membrane disorder does not exclude some specificity. Closely related drugs may have different molecular shapes and may be capable of forming hydrogen bonds with different orientations, affecting their ability to make membrane more fluid. Perhaps for this reason, there is a remarkable stereospecificity in the disordering effect of anesthetic steroids, chloralose, and long-chain alkenols. Some specificity is mediated by different membrane environments. The drug action may actually reverse from order to disorder on addition of cholesterol, but in other experimental systems cholesterol blocks a disordering effect, and we cannot yet explain the action of drugs in different biomembranes. Further, drugs may have differential solubilities in membranes of different composition. This cannot always be predicted from octanol:water partition coefficients because branched molecules are differentially excluded from structured bilayers. Charged drugs react quite differently with charged and neutral phospholipids and may have differential actions on the two sides of the bilayer because of the asymmetry of the phospholipid distribution. The deeper reaches of the membrane seem particularly sensitive to disordering, even by drugs that presumably reside near the surface. Thus, proteins whose midregions are sensitive to disordering may be especially disrupted by drugs. This is a new field of pharmacology, currently applied only to a small group of drugs. But an understanding of the physiocochemical actions of drugs in hydrophobic regions of cells will clearly be needed for full understanding of membrane-bound drug receptors, enzymes, and transport systems. This is just a beginning.

The Effects of Drugs on Membrane Fluidity

The effect of cholesterol on the acyl chain order of three glycerophosphocholines with 14, 16, and 18 carbons per acyl chain, namely, di(14:0)PC, di(16:0)PC, and di(18:0)PC, above the gel to liquid-crystalline phase transition temperature was investigated by using 2H nuclear magnetic resonance spectroscopy. Average acyl chain lengths were calculated from the segmental order parameters (Smol) for the sn-1 and the sn-2 chains in the absence of cholesterol and at 3:1, 2:1, and 1:1 mole ratios of phospholipid-cholesterol. The three binary mixtures of cholesterol with phosphatidylcholines are in the liquid-ordered (lo) phase. For all the three phosphatidylcholine-cholesterol systems, the distance from the carbonyl groups to the terminal methyl groups is shorter than the length of the cholesterol molecule. A molecular model for the lo phase consistent with these observations has in a statistical sense a part of each cholesterol molecule in one monolayer extending into the other monolayer. This results in a packing arrangement akin to that in interdigitated systems. On the basis of the effect of cholesterol on phospholipid acyl chain orientational order, it is suggested that the liquid-disordered (ld) phase at low cholesterol concentrations corresponds to a packing mode in which the cholesterol molecule spans the entire transbilayer hydrophobic region. A molecular mechanism is proposed in which increasing the concentration of cholesterol has the effect of stretching the acyl chains of phospholipids by increasing the population of trans conformers up to a stage where the hydrophobic length is considerably longer than the cholesterol molecule. Beyond this concentration, the partially interdigitated phase forms.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of phospholipid acyl chain order by cholesterol. A solid-state 2H nuclear magnetic resonance study.

Molecular order and dynamics of phosphatidylcholine bilayer membranes in the presence of cholesterol, ergosterol and lanosterol: a comparative study using 2H-, 13C- and 31p-NMR spectroscopy

George B. Zavoico

Papers

Effects of cholesterol on acyl chain dynamics in multilamellar vesicles of various phosphatidylcholines

Perturbation of egg phosphatidylcholine and dipalmitoylphosphatidylcholine multilamellar vesicles by n-alkanols. A fluorescent probe study.

Effects of n-Alkanols on the membrane fluidity of chick embryo heart microsomes