Free energies of urea and of thermal unfolding show that two tandem repeats of spectrin are thermodynamically more stable than a single repeat.

Free energies of both urea and thermal denaturation have been measured for three pairs of one- and two-repeat fragments, cloned in tandem from the cytoskeletal protein, alpha-spectrin, from chicken brain to ascertain whether one- and two-repeat fragments are equally stable. One- and two-repeat fragments of each pair were designed with the same N-terminus, whereas the C-terminus of the two-repeat fragment was 106 residues or the length of one repeat downstream from that of the one-repeat fragment. The averaged free energies of urea and thermal denaturation of the paired fragments, (R16)(00) and (R16R17)(00), (R16)(0+3) and (R16R17)(0+3), and (R16)(+8-4) and (R16R17)(+8-4) [subscripts represent the N- and C-terminal positions with "00" referring to the N- and C-termini defining a repeat according to X-ray crystal structures of two repeat fragments [Grum, V. L., Li, D., MacDonald, R. I., and Mondragón, A. (1999) Cell 98, 523-535] and "+" and "-" referring to positions upstream and downstream therefrom, respectively], increased from 3.7 +/- 0.4 kcal/mol for (R16)(00), 3.7 +/- 0.5 kcal/mol for (R16)(0+3), 4.4 +/- 0.4 kcal/mol for (R16)(+8-4), 6.2 +/- 0.6 kcal/mol for (R16R17)(+8-4), 8.3 +/- 0.4 kcal/mol for (R16R17)(00) to 9.9 +/- 1.0 kcal/mol for (R16R17)(0+3). Thus, the two-repeat fragment of each pair was significantly more thermodynamically stable than the single repeat by both urea and thermal denaturation. Differences in phasing among single repeats did not have the same effect as the same differences in phasing among two-repeat fragments. Addition of nine residues to the C-terminus of (R16R17)(00) yielded a free energy of unfolding of 7.9 +/- 0.8 kcal/mol, whereas addition of seven residues to the C-terminus of (R16)(+8-4) yielded a free energy of unfolding of 5.9 +/- 0.3 kcal/mol.

Structures of two repeats of spectrin suggest models of flexibility.

Spectrin is a vital component of the cytoskeleton, conferring flexibility on cells and providing a scaffold for a variety of proteins. It is composed of tandem, antiparallel coiled-coil repeats. We report four related crystal structures at 1.45 A, 2.0 A, 3.1 A, and 4.0 A resolution of two connected repeats of chicken brain alpha-spectrin. In all of the structures, the linker region between adjacent units is alpha-helical without breaks, kinks, or obvious boundaries. Two features observed in the structures are (1) conformational rearrangement in one repeat, resulting in movement of the position of a loop, and (2) varying degrees of bending at the linker region. These features form the basis of two different models of flexibility: a conformational rearrangement and a bending model. These models provide novel atomic details of spectrin flexibility.

Adaptive optoelectronic signal processor with metal-semiconductor-metal photodetector arrays.

Optoelectronic structures utilizing switched fiber delays and switched metal-semiconductor-metal photodetectors for signal processing over a 1.25-GHz bandwidth are presented. Fully tunable finite-impulse-response and infinite-impulse-response structures were synthesized with internal compensation for bias dependency of photodetectors. The application of such filter structures to the equalization of modal dispersion in a multimode fiber is also described.

Cytoskeletal protein binding kinetics at planar phospholipid membranes.

It has been hypothesized that nonspecific reversible binding of cytoskeletal proteins to lipids in cells may guide their binding to integral membrane anchor proteins. In a model system, we measured desorption rates k(off) (off-rates) of the erythrocyte cytoskeletal proteins spectrin and protein 4.1 labeled with carboxyfluorescein (CF), at two different compositions of planar phospholipid membranes (supported on glass), using the total internal reflection/fluorescence recovery after photobleaching (TIR/FRAP) technique. The lipid membranes consisted of either pure phosphatidylcholine (PC) or a 3:1 mixture of PC with phosphatidylserine (PS). In general, the off-rates were not single exponentials and were fit to a combination of fast, slow, and irreversible fractions, reported both separately and as a weighted average. By a variation of TIR/FRAP, we also measured equilibrium affinities (the ratio of surface-bound to bulk protein concentration) and thereby calculated on-rates, k(on). The average off-rate of CF-4.1 from PC/PS (approximately 0.008/s) is much slower than that from pure PC (approximately 1.7/s). Despite the consequent increase in equilibrium affinity at PC/PS, the on-rate at PC/PS is also substantially decreased (by a factor of 40) relative to that at pure PC. The simultaneous presence of (unlabeled) spectrin tends to substantially decrease the on-rate (and the affinity) of CF-4.1 at both membrane types. Similar experiments for CF-spectrin alone showed much less sensitivity to membrane type and generally faster off-rates than those exhibited by CF-4.1. However, when mixed with (unlabeled) 4.1, both the on-rate and off-rate of CF-spectrin decreased drastically at PC/PS (but not PC), leading to a somewhat increased affinity. Clearly, changes in affinity often involve countervailing changes in both on-rates and off-rates. In many of these studies, the effect of varying ionic strength and bulk concentrations was examined; it appears that the binding is an electrostatic attraction and is far from saturation at the concentrations employed. These results and the techniques implemented carry general implications for understanding the functional role of nonspecific protein binding to cellular lipid membranes.

Site-directed mutagenesis of either the highly conserved Trp-22 or the moderately conserved Trp-95 to a large, hydrophobic residue reduces the thermodynamic stability of a spectrin repeating unit.

As reported previously (MacDonald, R. I., Musacchio, A., Holmgren, R. A., and Saraste, M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 1299-1303), an unfolded peptide was obtained by site-directed mutagenesis of Trp-22 to Ala in the cloned, wild type 17th repeating unit (alpha17) of chicken brain alpha-spectrin. Trp occurs in position 22 of nearly all repeating units of spectrin. In the present study, Trp-22 was mutated to Phe or to Tyr to compare thermodynamic stabilities of urea-induced unfolding of alpha16 and mutants thereof. alpha16 was chosen for this study instead of alpha17, because alpha16 has two tryptophans, allowing urea-induced unfolding to be tracked by the fluorescence of the Trp remaining in each mutant peptide and by circular dichroism in the far UV. The free energies of unfolding of W22Y and W22F were 50% that of alpha16, showing that Trp-22 is crucial in stabilizing the triple helical bundle motif of the spectrin repeating unit. Mutation of the moderately conserved Trp-95 of alpha16 to Val, which occupies position 95 in alpha17, also yielded a peptide with 50% of the free energy of unfolding of alpha16. Thus, the thermodynamic stability of a given spectrin repeating unit may depend on both moderately and highly conserved tryptophans. Different structural roles of Trp-22 and Trp-95 in alpha16 are suggested by the slightly higher wavelength of maximum emission of Trp-22, the greater acrylamide quenching of Trp-95 than Trp-22, and the longer lifetime of Trp-95. For comparison with alpha16, urea-induced unfolding of spectrin dimer isolated from human red cells was monitored by far UV-CD and by tryptophan fluorescence. Thermodynamic parameters could not be rigorously derived for the stability of spectrin dimer because unfolding of spectrin dimer involved more than two states, unlike unfolding of cloned repeating units. However, the similar midpoints of CD-monitored denaturation curves of alpha16 and spectrin dimer, i. e. 2.7 and 3.2 M urea, respectively, indicate that investigation of cloned repeating units of spectrin can provide physiologically relevant information on these structures.

Design of phased-array wavelength division multiplexers using multimode interference couplers.

Novel designs for phased-array wavelength-division multiplexers based on self-imaging properties of multimode interference (MMI) couplers are presented. These devices, which operate on N equally spaced wavelength channels, consist of two MMI couplers connected by an array of N monomode waveguides. The MMI couplers function as power splitters/combiners, and the waveguide array is the dispersive element. The excellent characteristics of MMI couplers offer the possibility of designing small-size devices with low loss and with high uniformity among different channels. A general theoretical formulation for an N-channel multiplexer is presented, and a simple procedure for finding an optimum set of lengths for the array guides is given. We show that these multiplexers can function as N x N wavelength-selective interconnecting components. The simulated performance of three variations of a five-channel device, designed in a rib waveguide system, is given. It is demonstrated that sidelobes in the multiplexer spectral response can be suppressed by weighting the power samples in the array waveguides through appropriate design of a nonuniform MMI power splitter.

Invariant tryptophan at a shielded site promotes folding of the conformational unit of spectrin.

The tryptophan that is highly conserved among repeating structural units of spectrin is reported to promote the conformational stability of one such unit of chicken brain alpha-spectrin. Four constructs were inserted into pET vectors for overexpression in Escherichia coli of the following spectrin peptides: (i) two adjacent but separately expressed "conformationally phased" repeating units, R16 and R17, one of which (R17) contains a single tryptophan; (ii) a mutant, M17, of the single tryptophan-containing unit with alanine substituted for the tryptophan; and (iii) a conformationally unphased unit, 1617, composed of half of each of the phased units. Both the mutant unit and the unphased unit were much more readily digested by chymotrypsin and by elastase than the phased units and exhibited only 38% and 54% as much alpha-helical structure, respectively, as the phased units by their far UV CD spectra; 90 degrees light scattering measurements revealed the folded peptides to be predominantly monomeric in solution, whereas the unfolded, protease-sensitive peptides consisted of dimers and/or trimers. This trend was corroborated by their dynamic light scattering. Both the blue-shifted wavelength of maximal emission and the relative inaccessibility to acrylamide of the single tryptophan in the folded unit indicate that the invariant tryptophan occupies a site that is shielded from the aqueous phase.

Temperature and ionic effects on the interaction of erythroid spectrin with phosphatidylserine membranes.

Specific binding of human erythroid spectrin to large, unilamellar vesicles of bovine brain phosphatidylserine, made by an extrusion technique (LUVETs), has been measured and characterized by a new gel filtration assay. Vesicle-bound spectrin was separated from free spectrin by Sepharose CL-2B chromatography and detected by its intrinsic (tryptophan) or extrinsic (carboxyfluorescein) fluorescence. That the bound spectrin was not an aberrant, adhesive form was shown by the ability of a portion of free spectrin, which had not bound to PS LUVETs during a previous incubation, to bind during a subsequent incubation. Spectrin binding reached a plateau by 30 min of incubation at room temperature and at 37 degrees C. Binding increased from a low level below 31 degrees C to about twice as much as 37 degrees C and to 4-7 times as much between 40 and 43 degrees C. Similar results were obtained with LUVETs composed of DOPS but not PC. Triton treatment of PS LUVETs and spectrin after incubation of spectrin and vesicles at 40 and 43 degrees C but prior to chromatography on Sepharose CL-2B eliminated the bound spectrin peak, which thus did not consist of large aggregates of covalently associated spectrin. Binding isotherms fit by nonlinear regression gave an apparent Kd of 0.31 microM and an apparent maximum spectrin binding of 33 nM/mM PS at 25 degrees C, an apparent Kd of 0.35 microM and an apparent maximum spectrin binding of 40 nM/mM PS at 31 degrees C, and an apparent Kd of 3.4 microM and an apparent maximum spectrin binding of 113 nM/0.1 mM PS at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Band 4.1 enhances spectrin binding to phosphatidylserine vesicles.

Erythroid band 4.1 enhances the binding of erythroid spectrin to phosphatidylserine vesicles under conditions of ionic strength and at protein concentrations similar to those in the red cell. The extent of enhancement depends on the concentration of band 4.1; at 2 microM 4.1, spectrin binding increases approximately 10-fold (to 600 mumoles/mole lipid). The Kd is 0.5 microM, as measured by SDS-PAGE of protein-bound vesicles recovered by ficoll gradient centrifugation. The 4.1-enhanced binding of spectrin was also measured by a gel filtration assay. The electrostatic nature of the enhancement of spectrin binding is indicated by its dependence on the phosphatidylserine content of the vesicles.

Characteristics of spectrin-induced leakage of extruded, phosphatidylserine vesicles.

At neutral pH spectrin induces modest leakage of trapped calcein from reverse-phase or extruded, but not sonicated, vesicles composed of phosphatidylserine, but not phosphatidylcholine. The extent of leakage from extruded vesicles is not or is only slightly affected by magnesium ions at a physiological concentration or calcium ions at a greater than physiological concentration, respectively. In addition to accounting for several previously discrepant observations on the lytic effects of spectrin, these findings indicate that some proteins like spectrin may destabilize vesicles with low curvature more readily than vesicles of high curvature, in contrast to certain amphiphilic peptides. 60% less leakage is induced from phosphatidylserine vesicles by heat-denatured than by native spectrin. In contrast, both trypsin- and subtilisin-treated spectrins, if sufficiently digested, induce several-fold more leakage than undigested spectrin. Since spectrin prepared either by 1 M Tris dissociation of Triton-extracted cytoskeletons or by low ionic strength extraction of ghosts released the same amounts of calcein from vesicles of various compositions, these effects are unlikely to reflect artifacts of spectrin preparation. Furthermore, spectrin is unlikely to promote leakage in vivo, since vesicles composed of phosphatidylserine, cholesterol and/or phosphatidylethanolamine, which constitute the lipid composition of the inner monolayer of the red cell membrane, did not leak on addition of spectrin, whereas vesicles composed of phosphatidylserine and phosphatidylcholine, did leak in the presence of spectrin.

Small-volume extrusion apparatus for preparation of large, unilamellar vesicles.

The design and performance of a filter holder which enables convenient preparation of volumes of up to a milliliter of large, unilamellar vesicles formed by extrusion (LUVETs) from multilamellar vesicles (MLVs) are described. The filter holder provides for back-and-forth passage of the sample between two syringes, a design that minimizes filter blockage, eliminates the need to change filters during LUVET preparation and reduces preparation time to a few minutes. Replicas of slam-frozen LUVETs in the electron microscope are unilamellar and reasonably homogeneous with an average diameter close to the pore size of the filters used to extrude them. Extrusion per se does not destabilize the vesicles, which trapped a fluorescent dye only when they were disrupted on freeze-thawing and during the first extrusion when most of the MLVs were apparently converted to LUVETs.

Personal accounts of satisfying and unsatisfying nursing experiences as a needs assessment strategy.

This article describes the assessment process by which learning needs of a select group of nurses in a forensic psychiatric setting were identified and used for continuing education (CE) program planning. To determine whether a gap existed between what the nurses currently knew and what they needed to know in order to have a sense of accomplishment, their personal accounts of satisfying and unsatisfying nursing experiences were analyzed. The factors that influenced these experiences were categorized according to a theory of basic human needs, and learning needs were identified from those categories. Implications for the planning of content and methodology of CE programs for the study group were then developed.

Demonstration of a novel optical code-division multiple-access system at 800 megachips per second.

A novel optical code-division multiple-access system using Alberta codes and complementary correlation is demonstrated at 800 megachips per second. Completely passive optical multiplexing and demultiplexing is performed throughout the process. Complementary correlation permits code orthogonality because bipolar electrical signals are generated from unipolar optical ones. The correlator is implemented optoelectronically with an array of metal-semiconductor-metal photodiodes. We show that the bit-error-rate performance of the system is not degraded by interfering signals and that zero cross talk is nominally achieved. An unusual property of such systems is confirmed: The bit error rate on a channel can be reduced by the presence of an interfering channel if the two transmitters are not chip asynchronous.

Photodetector sensitivity control for weight setting in optoelectronic neural networks.

In optoelectronic neural processors weights are set optically and neural states electrically. Reversing this arrangement allows weights to be set by adjusting photodetector sensitivity.

Characteristics of self-quenching of the fluorescence of lipid-conjugated rhodamine in membranes.

Self- or concentration quenching of octadecylrhodamine B (C18-Rh) fluorescence increases linearly in egg phosphatidylcholine (PC) vesicles but exponentially in vesicles composed of egg PC:cholesterol, 1:1, as the probe concentration is raised to 10 mol%. Cholesterol-dependent enhancement of self-quenching also occurs when N-(lissamine-rhodamine-B-sulfonyl)dioleoylphosphatidylethanolamine is substituted for C18-Rh and resembles that in dipalmitoylphosphatidylcholine vesicles below, as opposed to above, the phase transition. These effects are not due to changes in dimer:monomer absorbance. Stern-Volmer plots indicate a dependence of quenching on nonfluorescent dimers both in the presence and absence of cholesterol. Decreases in fluorescence lifetimes with increasing probe concentration parallel decreases in residual fluorescence of C18-Rh with increasing probe concentration in PC and PC + cholesterol membranes, respectively. Decreases in the steady-state polarization of C18-Rh fluorescence as its concentration is raised to 10 mol% indicate energy transfer with emission between probe molecules in PC and to a lesser extent in PC + cholesterol membranes. The calculated R0 for 50% efficiency of energy transfer from excited state probe to monomer was 55-58 A and to dimer was 27 A. Since lateral diffusion of C18-Rh is probably too slow to permit collisional quenching during the lifetime of the probe, even if C18-Rh were concentrated in a separate phase, C18-Rh self-quenching appears to be due mainly to energy transfer without emission to nonfluorescent dimers.

Frequency domain optical reflectometer using a Gaas optoelectronic mixer.

A frequency domain optical reflectometer which uses a GaAs interdigital photodetector both for optical detection and for mixing is described. A simple receiver with a postdetection bandwidth of the order of 10 kHz suffices for distance resolution of 10 m with this technique, greatly simplifying the electronics by comparison with pulsed reflectometers or swept frequency reflectometers employing electronic mixing. With available lasers the instrument could detect discrete optical reflections of -57 dB at a distance of 1 km. It is intended for remote measurement of refractive index in fluids, using the Fresnel reflection from a square-cleaved fiber as the sensor.

Phosphatidylserine vesicle lysis by Sendai virus at low pH is not due to virus-vesicle fusion.

As a model of the fusion of Sendai virus with red cells, the interaction of the virus with phosphatidylserine (PS) vesicles at pH 5 was quantitated by the release of a trapped marker from target vesicles and by mixing of lipids of the virus and the vesicles. Release of the marker was measured as dequenching of calcein trapped at a self-quenched concentration and lipid mixing was measured as a decrease in energy transfer between fluorescent phospholipid analogs in the target membrane. At comparable virus:vesicle ratios both calcein release and lipid mixing were maximal at pH 5 and significantly reduced after trypsin, but not chymotrypsin, treatment. In contrast, these two effects differed in their PS dependence, time course, and temperature dependence, indicating that calcein release is not a consequence of the fusion of a permeable virus membrane with an impermeable target membrane. Vesicles composed of 25 to 100% PS released similar amounts of calcein, whereas fusion increased linearly as a function of PS content of the target vesicles. The half-time was 15 s for calcein release but 1.5 min for fusion. The temperature coefficient of fusion was at least three times greater than that of calcein release. These results indicate that calcein release at pH 5 may signify an interaction of the virus with PS target membranes which precedes but does not necessarily culminate in fusion, given too low a temperature or an inappropriate target membrane composition.

Membrane surface pressure can account for differential activities of membrane-penetrating molecules.

It is a common observation that cis-unsaturated and branched chain fatty acids, which are usually liquid, affect membrane function differently from saturated and trans-unsaturated fatty acids, which are usually solid. We also found that the former are much more potent than the latter in inhibiting viral hemolytic activity. A search for the origin of this difference revealed a correlation between inhibition and equilibrium surface pressure (the surface pressure at the air/water interface of a solution of the substance in question). Using a simple but rigorous thermodynamic analysis, we show that penetration of a lipid bilayer is correlated with equilibrium surface pressure of the penetrating molecule. We therefore conclude that an important reason for the difference in effects of liquid and solid fatty acids on membranes is the greater penetrability of the former relative to the latter. We suggest that attributing such effects to fluidity changes in the membrane should await demonstration of actual intramonolayer residence of the fatty acid in the membrane. The thermodynamic analysis is readily generalized and, in the absence of specific interactions between penetration and bilayer molecules, provides a convenient method for predicting membrane penetration by virtually any type of exogenous molecule.

Fully orthogonal optical-code multiplex for broadcasting.

A novel method of optical-code multiplex transmission from a central location is proposed. It has the advantages that the receivers can be configured to any channel quickly, the channels have in principle zero cross talk, and the bandwidth-expansion factors are less than for other optical-code-division multiple access arrangements. The proposed method is based on arrays of optoelectronic switching detectors that are at present under development for broadband matrix switching.

Energy transfer measurements of fusion between Sendai virus and vesicles corrected for decreased absorption of acceptor probe.

The fusion of Sendai virus at pH 4-7 with artificial lipid vesicles composed of phosphatidylserine or phosphatidylcholine was quantified by measuring fluorescence energy transfer from N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-phosphatidylethanolamine to N-(lissamine-rhodamine-B-sulfonyl)-phosphatidylethanolamine in the target membranes. About 60% of the phosphatidylserine vesicles and virus appeared to fuse at pH 4 and about 100% at pH 5. Fusion was much less under all other conditions. The apparent fusion at pH 4, however, was due to a decrease in absorption of the acceptor probe, instead of dilution of acceptor as a result of fusion of labeled vesicles with unlabeled virus. After correction for this fusion-independent effect of Sendai virus, the extent of fusion was only 4-20% at pH 4 but still 80-100% at pH 5. These findings paralleled the loss of hemagglutinating and hemolytic activities of the virus induced by incubation at pH 4 but not at pH 5. Vesicle-virus hybrids were observed with the electron microscope after incubation at pH 5 but not at pH 7. The assay of membrane fusion by fluorescence energy transfer can be misleading unless correction is made for changes in energy transfer due to fusion-independent effects.