Peter W. Holloway

Distribution analysis of membrane penetration of proteins by depth-dependent fluorescence quenching.

A new approach is presented to evaluate the depth-dependent quenching of the fluorescence of membrane-bound probes and integral proteins. By utilizing at least three quenchers of known and distinctly different depths, the following parameters can be recovered: most probable depth of the probe; dispersion of the depth distribution, which will depend on the size of probe and fluctuations in its position; and quenching efficiency, which is related to the exposure of a particular fluorophore to the lipid phase. The exposure of tryptophan residues in integral proteins can be quantitatively determined with respect to the model compound (tryptophan octyl ester). The proposed method was applied to the investigation of membrane complexes of the bee venom melittin and cytochrome b5.

Amino acid substitutions in the membrane-binding domain of cytochrome b5 alter its membrane-binding properties

The structure-function relationships of the 43-amino-acid membrane-binding domain of cytochrome b5 have been examined in two mutant forms of the protein. In one mutant, two tryptophans in the membrane-binding domain, at positions 108 and 112, were replaced by leucines, and in the second mutant, in addition, aspartic acid 103 was also replaced by leucine. The fluorescence emission spectra of the three proteins and their degree of quenching by brominated lipids indicate that the mutations are not producing major conformational changes or allowing a deeper degree of penetration of the domain into the bilayer. The hydrophobicities of the three proteins were compared, by determining strengths of self-association and membrane affinities, and it was found that the protein with two additional leucines was much less hydrophobic and the one with three additional leucines was much more hydrophobic than the native cytochrome. It appears that small changes in amino acid composition, which produce no gross changes in the structure of the membrane-binding domain, will nevertheless produce very large changes in the strengths of self- and membrane-association. These differences in self-association had profound effects on the times required for membrane-association to reach equilibrium.

Distribution of low density lipoprotein in the branch and non-branch regions of the aorta☆

Atherosclerosis occurs focally in branch segments of the artery. Understanding why these segments are more susceptible to the development of the disease is at the root of understanding atherogenesis. We investigated accumulation of low density lipoprotein (LDL) in the branch and non-branch regions of the aorta to determine why the disease develops in branch regions. Abdominal aortas and their major branches were harvested from 36 rabbits. Rabbit LDL was prepared from whole blood and radiolabeled with 125I. The aorta was incubated with radiolabeled LDL in the lumen at 37 degrees C, under intraluminal pressure of 2-3 mmHg, for 1 h. Disks of 1.8 mm diameter were punched from the branch and non-branch regions of the aorta, cryosectioned and the sections counted in a gamma counter. Protein bound radioactivity was determined by TCA precipitation. LDL accumulation was highest towards the aortic intima and declined sharply towards the media. LDL accumulation at any given depth was higher in the branch than non-branch region. LDL accumulation in the intimal-medial sections was 87% higher in the branch than non-branch region. Total LDL accumulation in the branch was almost twice that in the non-branch region. Mean LDL accumulation was also greater in the branch than non-branch region. The aorta was significantly thicker at the branch. LDL distribution profiles indicate that LDL is present in a greater concentration and over a greater depth in the branch than non-branch region. The tendency of the branch region to accumulate LDL in greater amounts may explain its susceptibility to atherosclerotic lesion development.

Binding of cytochrome b5 to cholesterol-containing phosphatidylcholine vesicles

Abstract Cytochrome b 5 was found to bind readily to sonicated vesicles containing as much as 0.8 mol cholesterol per mol egg phosphatidylcholine. This observation conflicts with the suggestion of Enomoto and Sato ((1977) Biochim. Biophys. Acta 466, 136–147) that cholesterol prevents binding of this protein to erythrocyte membranes.

Effect of Protein Aggregation in the Aqueous Phase on the Binding of Membrane Proteins to Membranes

Analysis of the binding of hydrophobic peptides or proteins to membranes generally assumes that the solute is monomeric in both the aqueous phase and the membrane. Simulations were performed to examine the effect of solute self-association in the aqueous phase on the binding of monomeric solute to lipid vesicles. Aggregation lowered the initial concentration of monomeric solute, which was then maintained at a relatively constant value at the expense of the aggregated solute, as the lipid concentration was increased. The resultant binding isotherm has a more linear initial portion rather than the classic hyperbolic shape. Although this shape is diagnostic of solute self-association in the aqueous phase, various combinations of values for the membrane partition coefficient and the solute self-association constant will generate similar isotherms. Data for cytochrome b5 were analyzed and, when the self-association constant was estimated by gel filtration, a unique value for the membrane partition coefficient was obtained. Thus, to obtain a true partition coefficient the state of the solute in the aqueous phase must be known. If the concentration of the monomeric solute species in the aqueous phase can be independently determined, then, even with heterogeneous aggregates, the true partition coefficient can be obtained.

The Interaction of Cytochrome b5 with Lipid Vesicles

Cytochrome b5 is a well-characterized intrinsic membrane protein found in the endoplasmic reticulum of liver cells and perhaps also in other intracellular membranes (1, 2). The protein can be isolated by detergent extraction and the amino acid sequence of cytochrome b5 from several animal species has been published (3). The isolated detergent and lipid free protein binds rapidly and completely to preformed phosphatidylcholine (PC) vesicles and other membranes. In investigating the binding properties of the protein we observed that the protein would exchange rapidly between PC vesicles (4). Studies of the binding and exchange have been facilitated by the tryptophan fluorescence of the protein, which increases up to two-fold upon binding to PC vesicles. It should be noted that, as reported (5), the kinetics of fluorescence enhancement seen with the protein are biphasic. The slow rate (t1/2= 30 s) is due to dissociation of octomeric protein to monomer (little fluorescence change) followed by the rapid fluorescence enhancement due to binding. The enhancement kinetics can be made monophasic (rapid) by use of monomeric protein (isolated by gel filtration). This preparation is used in all our studies. A fluorescence technique has also been used to monitor continuously exchange of protein between vesicles. For these studies we have used brominated lipid vesicles. The bromolipid, made by bromination of an octadecenoic acid (either A9 or A6) followed by coupling of the dibromide to 1 -palmitoylglycerophosphorylcholine, forms stable vesicles of similar size to those of I-palmitoyl-2-oleoyl glycerophosphorylcholine (POPC). The vesicles made from lipid containing the 9, 10 dibromide (9, 10 BrPC) show no phase transition between 50 and 500 by scanning calorimetry. Addition of 9, 10 BrPC vesicles to cytochrome b5 (b5) results in immediate quenching of fluorescence (to 50% of the original value). Subsequent addition of POPC vesicles results in a slow enhancement of fluorescence (t1/2 c 30 min). Addition in the reverse order (POPC then 9,10 BrPC) produces an immediate enhancement, followed by slow quenching. Detailed kinetic analysis of these data and the observation that dilution had no effect on the fractional rates of b5 exchange lead us to conclude that exchange proceeds via transfer of b5 through the aqueous phase rather than by collision of donor and acceptor vesicles (4). Confirmation of the exchange of the protein from the donor to the acceptor vesicle is made by separating the dense 9, 10

Modulation of membrane composition of swine vascular smooth muscle cells by homologous lipoproteins in culture

Swine vascular smooth muscle cells were exposed to homologous low-density or high-density lipoprotein fractions for 24 h. Total cell membranes were isolated from the post-nuclear supernatant of the cell homogenates, fractionated by sucrose denisty gradient centrifugation and characterized by enzyme assays. The membrane fraction with the lowest density was enriched in plasma membrane marker enzymes. Cholesterol analysis showed that cells exposed to low-density lipoprotein had higher cholesterol-to-protein ratios in total cells, total cell membranes and individual membrane fractions than had the cells exposed to high-density lipoproteins. Cholesterol-to-phospholipid ratios of the plasma membrane-enriched fraction from cells exposed to low-density lipoprotein were higher than the same membrane fraction of cells exposed to high-density lipoprotein. Studies with iodinated lipoproteins showed that these compositional changes could not be due to lipoprotein contamination. Membrane microviscosity was determined by fluorescence depolarization with diphenylhextriene and the microviscosity of the plasma membrane-enriched fraction was different in the cells exposed to the two different lipoprotein fractions. This difference in membrane microviscosity was significant only when the medium cholesterol content was 40 micrograms per ml or greater; cells exposed to low-density lipoprotein gave membranes with higher microviscosity. These results demonstrate that the properties of vascular smooth muscle cell membranes are influcenced by exposure of the cells to homologous lipoprotein fractions.

Stopped-Flow Fluorescence Studies of the Interaction of a Mutant Form of Cytochrome b5 with Lipid Vesicles

Cytochrome b5 binds spontaneously to lipid vescles and also self-associates in aqueous solution. Two mutant proteins have been generated, one has a self-association constant which is less than that of the native protein, while the other has a larger self-association constant. All three proteins have Trp in the membrane-binding domain but as aqueous solutions of these proteins contain differing amounts of monomeric protein, the kinetics of fluorescence enhancement, when the proteins are mixed with lipid vesicles, are complex. Similar complex kinetics are seen when the Trp are quenched by the addition of bromolipid vesicles. The mutant which has Trp 108 and 112 both replaced by Leu does not self-associate and shows monoexponential stopped-flow fluorescence kinetics. Identical rate constants are seen with this mutant for fluorescence enhancement by POPC and fluorescence quenching by three bromolipids with bromines at the 6,7-, 9,10-, and 11,12-positions of thesn-2 acyl chain. This rate constant is only 1% of the calculated collisional rate constant and it is suggested that the reduced rate is caused by a reduction in the number of productive collisions rather than by a slow rate of penetration of the membrane-binding domain into the bilayer.

Frequency-domain fluorescence of mutant cytochrome b5

Cytochrome b5, isolated by detergent extraction from rabbit liver, has been extensively studied by fluorescence techniques, however, its fluorescence properties are complicated by the presence of three tryptophans in the membrane-binding domain. This protein has now been expressed in E. coli and a mutant form has been isolated which contains only one tryptophan (Trp-109) in the membrane-binding domain. The mutant protein does differ from the native protein in several of its spectroscopic properties, but it interacts with lipids in a similar way and shows the same functional activity. The availability of a mutant cytochrome b5 with a single Trp in the nonpolar domain enables several frequency-domain experiments: to estimate the distribution of distances between Trp-109 and the heme; to study the incorporation of the protein into the membrane; to study the quenching of the tryptophan fluorescence by lipids with a brominated acyl chain; and to analyze the shape of the lifetime distribution.

FTIR Analysis of Cytochrome bs in H2O and D2O

The application of infrared spectroscopy to biological samples has been made possible largely by the advent of FTIR. The advances in instrumentation which have allowed this application are well documented, however, there are still some barriers to the full utilization of this technique for biological samples such as proteins. The general objectives are to obtain spectra in the 1700 - 1600 cm -1 region, where the Amide I band is located. This broad, generally featureless, band is a complex composite of bands which are characteristic of specific types of secondary structure in the protein 1. The problems to be overcome fall into several categories. The need to work in aqueous solutions, where water has a very large absorption band at 1650 cm4, has been circumvented to a large extent by working in D20, although it is still necessary to employ short path length cells, as the absorbance of the H-O-D overlaps both the Amide I and Amide II regions. In spite of the similarity of D20 and H2O, there are still advantages to obtaining spectra in both solvents as in D20 the accessible N-H groups in the peptide bonds will undergo H->D exchange. A comparison between spectra obtained in the two solvents will therefore give information on solvent accessibility of the peptide bonds in the different types of secondary structure and will also assist in assigning bands to particular secondary structures. Coupled with this requirement of short path length cells is the tendency of proteins to denature on surfaces. This requires that demountable cells be used and these cells, especially with 6 - 50 μm spacers, have a variability in path length from sample to sample, and especially from sample to the solvent blank which makes subtraction of the solvent blank very difficult. This problem of solvent subtraction is most acute when H2O is the solvent as here the high solvent absorbance is coupled with the smaller, and more variable, path length. Once the sample spectrum minus the solvent has been obtained the broad Amide I band has to be subjected to some type of enhancement procedure, usually Fourier self-deconvolution2, in order to resolve the bands due to particular types of protein secondary structure. Both the solvent subtraction and the deconvolution are somewhat subjective and open to criticism. In this report we demonstrate that reproducible deconvolved spectra can be obtained in both D20 and H2O.