Lateral diffusion coefficients in membranes measured by resonance energy transfer and a new algorithm for diffusion in two dimensions

We describe measurements of lateral diffusion in membranes using resonance energy transfer. The donor was a rhenium (Re) metal-ligand complex lipid, which displays a donor decay time near 3 micros. The long donor lifetime resulted in an ability to measure lateral diffusion coefficient below 10(-8) cm(2)/s. The donor decay data were analyzed using a new numerical algorithm for calculation of resonance energy transfer for donors and acceptors randomly distributed in two dimensions. An analytical solution to the diffusion equation in two dimensions is not known, so the equation was solved by the relaxation method in Laplace space. This algorithm allows the donor decay in the absence of energy transfer to be multiexponential. The simulations show that mutual lateral diffusion coefficients of the donor and acceptor on the order of 10(-8) cm(2)/s are readily recovered from the frequency-domain data with donor decay times on the microsecond timescale. Importantly, the lateral diffusion coefficients and acceptor concentrations can be recovered independently despite correlation between these parameters. This algorithm was tested and verified using the donor decays of a long lifetime rhenium lipid donor and a Texas red-lipid acceptor. Lateral diffusion coefficients ranged from 4.4 x 10(-9) cm(2)/s in 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPG) at 10 degrees C to 1.7 x 10(-7) cm(2)/s in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) at 35 degrees C. These results demonstrated the possibility of direct measurements of lateral diffusion coefficients using microsecond decay time luminophores.     

Effect of disordered hemes on energy transfer rates between tryptophans and heme in myoglobin.

Our recent linear dichroism study of heme transitions (Gryczynski, Z., E. Bucci, and J. Kusba. 1993. Photochem. Photobiology. in press) indicate that heme cannot be considered a planar oscillator when it acts as an acceptor of radiationless excitation energy transfer from tryptophan. The linear nature of the heme absorption transition moment in the near-UV region implies a strong dependence of the transfer rate factors on the relative angular position of the heme and tryptophan, i.e., on the kappa 2 orientation parameter of the Förster equation. Using the atomic coordinates of SW myoglobin we have estimated the variation of kappa 2 parameter as a function of the heme absorption transition moment direction. The simulations proved that transfer is very efficient and anticipates lifetimes in the picosecond range. Also, they showed that transfer is very sensitive to rotations of the heme around its alpha-gamma-meso-axis, which may reduce the efficiency of transfer to almost zero values, producing lifetimes very similar to those of free tryptophan, in the nanosecond range. Comparisons between the lifetime values reported in the literature and those here estimated suggest that natural heme disorder, in which heme is rotated 180 degrees around its meso axis, is at the origin of the nanosecond lifetimes found in myoglobin systems.     

On the taste of stereoisomeric cyclic dipeptides containing a proline residue

An investigation of the taste character and intensity of sixproline-containing cylic dipeptides demonstrates that thesecompounds are differentiated largley on the basis of their chemicalconfiguration. ^x/ Permanent address: Technical University, 80–952 Gdask,Poland     

To the problem of biologically active conformation of enkephalin

Summary A semi-rigid structural analog of [Leu⁵] enkephalin, possessing the azo-bridge between Tyr¹ and Phe⁴ residues, was synthesized, along with two other linear enkephalin analogs: [4′-amino Phe⁴] enkephalin and [4′hydroxyphenyl/-azo Phe⁴] enkephalin. The results of the determination of the analgesic activity of the synthesized compounds suggest that the biologically active conformation of the enkephalin molecule should be such that both aromatic rings, Tyr¹ and Phe⁴, are situated in close proximity.     

Directional surface plasmon-coupled emission from a 3 nm green fluorescent protein monolayer

High-sensitivity detection schemes are of great interest for a number of applications. Unfortunately, such schemes are usually high-cost. We demonstrate a low-cost approach to a high-sensitivity detection scheme based on surface plasmon-coupled emission (SPCE). The SPCE of a monomolecular layer of green fluorescent protein (GFP) is reported here. The protein was electrostatically attached to a thin, SiO(2)-protected silver film deposited on a quartz substrate. The visible, directional emission of GFP was observed at a sharp, well-defined angle of 47.5 degrees from the normal to the coupling prism, and the spectrum corresponded to that of GFP. The SPCE resulting from the reverse Kretschmann configuration showed a 12-fold enhancement over the free space fluorescence. The directional emission was 97% p-polarized. The directionality and high polarization can be coupled with the intrinsic spectral resolution of SPCE to be used in the design miniaturized spectrofluorometers. The observation of SPCE in the visible region of the spectrum from a monolayer of protein opens up new possibilities in protein-based sensing.     

Spectroscopic characterization of the EF-hand domain of phospholipase C delta1: identification of a lipid interacting domain

The interaction of the isolated EF-hand domain of phospholipase C delta1 with arachidonic acid (AA) was characterized using circular dichroism (CD) and fluorescence spectroscopy. The far-UV CD spectral changes indicate that AA binds to the EF domain. The near-UV CD spectra suggest that the orientations of aromatic residues in the peptide are affected when AA binds to the protein. The fluorescence of the single intrinsic tryptophan located in EF1 was enhanced by the addition of dodecylmaltoside (DDM) and AA suggesting that this region of the protein is involved in hydrophobic interactions. In the presence of a low concentration of DDM it was found that AA induced a change in fluorescence resonance energy transfer, which is indicative of a conformational change. The lipid induced conformational change may play a role in calcium binding because the isolated EF-hand domain did not bind Ca2+ in the absence of lipids, but Ca2+-dependent changes in the intrinsic tryptophan emission were observed when free fatty acids were present. These studies identify specific EF-hand domains as allosteric regulatory domains that require hydrophobic ligands such as lipids.     

Voltammetry as a virtual potentiometric sensor in modelling of a metal-ligand system and refinement of stability constants. Part 4. An electrochemical study of NiII complexes with methylene diphosphonic acid

The Ni(II)-MDP-OH system (MDP=methylene diphosphonic acid) and stability constants of complexes formed at ionic strength 0.15M at 298K were established by direct current polarography (DCP) and glass electrode potentiometry (GEP). The final M-L-OH model could only be arrived to by employing recent concept of virtual potentiometry (VP). VP-data were generated from non-equilibrium and dynamic DC polarographic technique. The VP and GEP data were refined simultaneously by software dedicated to potentiometric studies of metal complexes. Species distribution diagrams that were generated for different experimental conditions employed in this work assisted in making the final choice regarding the metal-ligand model. The model established contains ML, ML(2), ML(OH) and ML(OH)(2) with stability constants, as logbeta, 7.94+/-0.02, 13.75+/-0.02, 12.04 (fixed value), and 16.75+/-0.05, respectively. It has been demonstrated that virtual potential must be used in modelling operations (predictions of species formed) when a polarographic signal decreases significantly due to the formation of polarographically inactive species (or formation of inert complexes). The linear free energy relationships that included stability constant logK(1) for Ni(II)-MDP established in this work together with other available data were used to predict logK(1) values for Sm(III) and Ho(III) with MDP. The logK(1) values for Sm(III)-MDP and Ho(III)-MDP were estimated to be 9.65+/-0.10 and 9.85+/-0.10, respectively.     

A protein biosensor for lactate

Blood lactate is a clinically valuable diagnostic indicator. In this preliminary report we describe a protein biosensor for L-lactate based on beef heart lactate dehydrogenase (LDH). LDH was noncovalently labeled with 8-anilino-1-naphthalene sulfonic acid (ANS). The ANS-labeled LDH displayed an approximately 40% decrease in emission intensity upon binding lactate. This decrease can be used to measure the lactate concentration. The ANS-labeled LDH was further utilized in a new sensing format, polarization sensing, which is suitable for miniaturization to a point-of-care lactate monitor. However, temporal instability of beef heart LDH indicates the need for further protein engineering prior to development of a more robust lactate-sensing protein.     

Evidence for pre- and post-power stroke of cross-bridges of contracting skeletal myofibrils

We examined the orientational fluctuations of a small number of myosin molecules (approximately three) in working skeletal muscle myofibrils. Myosin light chain 1 (LC1) was labeled with a fluorescent dye and exchanged with the native LC1 of skeletal muscle myofibrils cross-linked with 1-ethyl-3-[3(dimethylamino) propyl] carbodiimide to prevent shortening. We observed a small volume within the A-band (∼10⁻¹⁵ L) by confocal microscopy, and measured cyclic fluctuations in the orientation of the myosin neck (containing LC1) by recording the parallel and perpendicular components of fluorescent light emitted by the fluorescently labeled myosin LC1. Histograms of orientational fluctuations from fluorescent molecules in rigor were represented by a single Gaussian distribution. In contrast, histograms from contracting muscles were best fit by at least two Gaussians. These results provide direct evidence that cross-bridges in working skeletal muscle assume two distinct conformations, presumably corresponding to the pre- and post-power-stroke states.     

Enkephalin analogs modified in the aromatic ring of the N-terminal tyrosine residue

Two analogs of Leu⁵-enkephalin, (1-O-methyltyrosine,5-leucine)-enkephalin and [1-(3′-amino)-tyrosine,5-leucine]-enkephalin, were synthesized by classical methods. Both analogs show high biological potency after injection into the lateral brain ventricle of the rat. In both cases substitution of the Tyr residue of enkephalin leads to a pronounced prolongation of analgesic action, as compared with the unsubstituted peptide.