Fluidity Parameters of Lipid Regions Determined by Fluorescence Polarization

The atomic models of the complex between rabbit skeletal muscle actin and bovine pancreatic deoxyribonuclease I both in the ATP and ADP forms have been determined by X-ray analysis at an effective resolution of 2.8 A and 3A, respectively. The two structures are very similar. The actin molecule consists of two domains which can be further subdivided into two subdomains. ADP or ATP is located in the cleft between the domains with a calcium ion bound to the beta- or beta- and gamma-phosphates, respectively. The motif of a five-stranded beta sheet consisting of a beta meander and a right handed beta alpha beta unit appears in each domain suggesting that gene duplication might have occurred. These sheets have the same topology as that found in hexokinase.

Atomic structure of the actin:DNase I complex.

The effect of iodide on the tryptophyl fluorescence of model compounds and of lysozyme was studied in order to evaluate the factors that determine the use of iodide as a selective quencher of the fluorescence of tryptophyl side chains of proteins exposed to solvent. The results with the model compounds indicate the involvement of a collisional quenching mechanism due to the agreement with the Stern-Volmer law and the proportionality of the quenching constant with To7 for indole-3-acetamide. Bimolecular rate constants, k a , calculated from measured quenching constants using available lifetime data are equal to, greater than, or less than 4-6 X lo9 M-' sec-l for uncharged, positively charged, and negaI n a preliminary study it was shown that a large fraction of the tryptophyi fluorescence of lysozyme in aqueous solution was quenched by low concentrations of iodide ion (Lehrer, lY67). It was concluded from a study of the magnitude of the quenching of fluorescence and the character of the difference fluorescence spectrum produced in the presence and absence of substrate that the fluorescence of tryptophyls exposed to solvent and located in the substrate binding site was preferentially quenched by iodide. It appeared that this technique, which can be called solute perturbation of protein fluorescence, could be used as a probe of fluorophor exposure in proteins in a manner analogous to the technique of solvent perturbation of protein absorption (Herskovits and Laskowski, 1960; Laskowski, 1966). * From the Department of Muscle Research, Boston Biomedical Research Institute, Boston, Massachusetts 021 14, and from the Department of Neurology, Harvard Medical School, Boston, Massuchusetts 02115. Receired April 22, 1971. This work was supported by grants from the National Institutes of Health (AM 11677 and HE 0581 1) and the iMass'ichuserts Heart Association (516). tively charged tryptophyl compounds, respectively. A modified version of the Stern-Volmer law was calculated for a fluorophor population with different quantum yields and quenching constants. This formulation allows the calculation of the effective quenching constant from the intercept and the slope at low iodide concentration of a F o ] M cs. l/(I-) plot. Data obtained for lysozyme indicate that for the native protein about one-half the tryptophyl fluorescence is accessible at pH 5.3 whereas all of the tryptophyl fluorescence is accessible in 6 M G d n . HCI. Information regarding the presence of charged groups near tryptophyl side chains was obtained for lysozyme by studying the dependence of the quenching on pH. More recently, studies by other workers have ~ised bromate (Winkler, 1969) and iodide (Arrio er al., 1970) to quench extrinsic fluorescence (Teale and Badley, 1970). Oxygen has also been used as a quencher of pyrenebutyric acid bound to proteins (Vaughan and Weber, 1970). Burstein (1968a) has also independently studied the quenching of tryptophyl fluorescence in model compounds by iodide. In order to learn more about the quenching mechanism and the factors which determine fluorophor exposure, various tryptophyl model compounds and a model protein, lysozyme. were used in the present study. The results of the model compound study provide evidence for a mechanism that follows the classical Stern-Volmer law (1919), predominantly involving collisional quenching, and illustrate the importance of local charge and solvent viscosity. The quenching of lysozyme fluorescence by iodide also appears to follow a similar mechanism because of the agreement obtained with a inodified version of the Stern-Volmer law which was calculated for a heterogeneous distribution of fluorophors in a protein. Effective Stern-Volmer quenching constants and values for the fractional accessible fluorescence were obtained for lyso3254 B I O C H E M I S T R Y , V O L . 1 0 , N O . 1 7 , 1 9 7 1 I O D I D E Q U E N C H I N G O F P R O T E I N F L U O R E S C E N C E zyme in 6 M Gdn.HCI, 'S M urea, and in aqueous solution at different pH's using the modified Stern--Volmer law. Values obtained are consistent with information regarding accessibility obtained by other methods. Experimental Section Muteriais. The following high-purity compounds were used as obtained from Mann Research Laboratories, New York, N. Y. : indole-3-acetic acid, indole-3-propionic acid, indole-3-butyric acid, indole-3-acetamide, N-Ac-L-TrpNH?, L-TrpOEt, Gdn . HCI, and urea. L-Trp (Cyclo Chemical Corp., Los Angeles), KI , Na&03, citric acid, and NaCl (Fisher Scientific Co., Freehold, N. J.) were all of high purity and used as obtained. Indole (Fisher) and skatole (3-methylindole) (Mann) were recrystallized from methanol containing Norit A (Matheson Coleman & Bell, Rutherford, N. J.). Hepes buffer was used as obtained from Calbiochem (Los Angeles). Poly(Glug9Trp1) and poly(Lysg7Trp3) were high molecular weight random sequence copolymers kindly supplied by Dr. G. Fasman. Lysozyme from two different sources were used (twice crystallized from Worthington Biochemical Corp., Freehold, N. J., and six-times crystallized from Miles Laboratories, Elkhart, Ind.). Both preparations gave similar results. Ac3Glcn was kindly supplied by Dr. J. Rupley and glycol chitin was obtained from Miles Laboratories. Methods. Quenching measurements at constant pH were made on five solutions of a given material containing increasing amounts of K I (0-0.2 M). These were prepared by diluting stock solutions of the model compound, of KI, of NaC1, and of buffer, into volumetric flasks. NaCl was used to keep the ionic strength constant. Stock solutions of the indole compounds were used within a few days of preparation and kept in the dark at 0-5" overnight. A small amount of SO3?(ca. M) was added to the iodide solution to prevent 1 3 formation. This was necessary because Isabsorbs in the wavelength region of tryptophyl fluorescence (filter effect) and because of possible chemical reaction. The solutions were equilibrated at 25 O before the measurements. Stock solutions of lysozyme were routinely filtered through a Millipore filter (HAWP 0.45 p ) before use. pH titrations were performed in the absence and presence of iodide by adding small quantities of 0.5 M HC1 to the solution in the cuvet, which contained 2 mM Hepes and 2 mM citrate, originally pH 8, then measuring the pH and fluorescence. pH was measured with a Radiometer PHM4c meter standardized at pH 4 and 7. Fluorescence spectra and intensities were measured by exciting a t 280 nm or longer. In most cases no corrections for iodide absorption were necessary. The fluorescence of a reference (usually the 0.2 M NaC1-0.0 M K I solution) was measured just before measuring the fluorescence of each solution in order t o correct for any exciting lamp fluctuation. Fluorescence measurements were made with either an Aminco-Bowman spectrofluorometer or an instrument that employs two Jarrell-Ash 0.25-m monochromators, an EM1 9601 B photomultiplier, and either a high-pressure 200-W mercury lamp or a 150-W high-pressure xenon lamp. Low temperatures were obtained with a refrigerated water circulator attached to the sample housing. The temperature was measured by inserting a calibrated thermistor into the sample solution. Abbreviations used are: Gdn . HCI, guanidine hydrochloride; Trp, tryptophyl or tryptophan; Hepes, N-2-hydroxyethylpiperazine-N'2-ethanesulfonic acid ; Ac3GIcii. tri-N-acetyl-D-glucosamirie. The activity of lysozyme was determined by the method of Hamaguchi et a/ . (1960). The decrease in viscosity with time caused by hydrolysis of glycol chitin (2 mg/ml) by lysozyme (0.02 mg/ml) in the presence of 0.2 M NaCl or 0.2 M KI in 2 m M citrate (pH 5.5) is the basis of this method. The specific viscosity of glycol chitin solutions in Cannon viscometers at 25" was measured with time after a small volume of lysozyme was added. The slope of the approximately linear viscosity decrease between 1 and 10 min was used as a measure of activity. The optical rotatory dispersion and circular dichroism spectra of lysozyme (0.95 mg/ml) in 0.2 M NaCl or in 0.2 M KI , 2 mM citrate (pH 5.2) were measured in a 1-cm cell with a Jasco spectropolarimeter. The absorbance of Iprevented measurements below 265 nm. Difference spectra were either measured with a Cary 15 or a Beckman DK spectrophotometer using mixing cells (Pyrocell, Inc., N. Y . ) . The total absorption over the wavelengths scanned was always below 2.2. The low-temperature studies were performed with a Beckman D K using a refrigerated sample holder.

Solute perturbation of protein fluorescence. The quenching of the tryptophyl fluorescence of model compounds and of lysozyme by iodide ion.

Fluorescence quenching studies with proteins.

Publisher Summary This chapter describes many of the techniques employed to study water–macromolecules interactions, their general usefulness, points out areas of mutual support and contradiction. The chapter also presents hydration of other macromolecules and the hydration of small molecules. Four broadly based approaches to operational definitions of hydration include—(1) preferential hydration, (2) hydrodynamic hydration, (3) structural hydration, and (4) low temperature hydration. The use of high resolution X-ray and neutron diffraction data on protein crystals provides an independent approach for estimation of hydration. The chapter presents several proposals for predicting protein hydration based on the amino acid composition of the protein; however, the two main questions concerned include—whether ionic groups are more hydrated than other polar groups, and whether the amide and peptide functions are hydrated or not. Low pH, and to a much smaller degree, high pH should dehydrate proteins. Intermolecular association via ionic mechanisms should be strongly dehydrating; whereas hydrophobic interactions should not have much effect on hydration levels. Although, there is a presumption that both the enthalpy and the entropy are negative for the process of adding water molecules to isolated proteins, which are maintained in their native conformations, but there is no unequivocal evidence for this.

Hydration of Proteins and Polypeptides

Human apolipoprotein C-I (apo C-I) in solution, in monomeric and oligomeric form, and in micellar complexes with dimyristoylphosphatidylcholine (DMPC), below and above the phase transition temperature of DMPC, was investigated with steady-state and time-resolved fluorescence methods. The environment of the Trp residue of apo C-I, in each physical state, was evaluated from fluorescence spectra and their changes upon KI quenching. Rotational correlation times of Trp residues were obtained from fluorescence anisotropy decay measurements. Static fluorescence anisotropy was determined as a function of temperature for the Trp residues of apo C-I in all physical states and for diphenylhexatriene dissolved in apo C-I X DMPC complexes. It was found that the Trp residues of apo C-I in solution are exposed from 75 to 88% to the aqueous medium, depending on the state of self-association. On the other hand, the Trp residues in apo C-I X DMPC complexes are only 42-45% exposed to KI quenching through an environment distinct from water. Apolipoprotein C-I in all its physical forms had two rotational correlation times associated with Trp motions: a longer one dependent on the size and flexibility of the entire particle and a very short one in the range from 0.2 to 0.4 ns. The later correlation times correspond to local Trp residue motions. These Trp motions were not significantly affected by a transition from the gel to the liquid-crystalline state of the lipid in apo C-I X DMPC complexes, suggesting that there is no coupling between the local motions of lipids and those of Trp side chains of apo C-I.

Nanosecond rotational motions of apolipoprotein C-I in solution and in complexes with dimyristoylphosphatidylcholine

We present evidence that liposomes (composed of egg yolk L-alpha-phosphatidylcholine/phosphatidylethanolamine/cholesterol, in a molar ratio of 4:5:1) fuse with isolated mouse liver nuclei at low pH. Using the resonance energy-transfer assay, we determined the rate and extent of liposome and nuclear membrane lipid mixing. Fusion was substantial when the pH was below 5. The half-time of lipid mixing decreased by acidification of the solvent, reaching about 2 min at pH 4.5. In order to study the transport of the liposome-aqueous contents to the interior of the nuclei during the process, we developed fluorescence assays in which fluorescein isothiocyanate labeled dextrans of 150 kDa molecular mass (FITC-D150) were encapsulated in liposomes. These liposomes also included in their bilayers the fluorescent lipid N-tetramethylrhodamine-L-alpha-dipalmitoylphosphatidylethanolamine (N-Rh-DPPE). After incubation of these liposomes with mouse liver nuclei (pH 4.5, 37 degrees C, 30 min), we measured the fluorescence spectra of a suspension of washed nuclei and of nuclei treated by the detergent Triton X-100 (membrane-denuded nuclei). These Triton X-100 treated nuclei had no N-Rh-DPPE fluorescence while they showed a FITC-D150 fluorescence which amounted to 20% of that of the intact nuclei. In another assay, a laser beam was focused on single nuclei by a microscope epiexcitation device. The variation of the N-Rh-DPPE and FITC-D150 fluorescence with the nuclear radius was determined with the microphotometric attachment of the microscope.(ABSTRACT TRUNCATED AT 250 WORDS)

Resonance energy-transfer and fluorescence intensity studies of the transport of liposome-encapsulated molecules into isolated mouse liver nuclei.

Nanosecond-pulse fluorimetry of wheat germ agglutinin is analyzed as a function of both excitation and emission wavelengths. When excited at 280 nm, wheat germ agglutinin fluorescence exhibited three lifetimes: one corresponding to the tyrosine residues as a whole and two others corresponding to the tryptophyl emission. The tyrosine contribution to the emission spectrum deduced from this method was in good agreement with that reported previously in steady-state fluorescence experiments [Privat, J.P. and Monsigny, M. (1975) Eur. J. Biochem. 60, 555-567]. The fluorescence decay of each tryptophan residue was not a single exponential function when wheat germ agglutinin was excited at 295 nm. This could be related to the microenvironment of the indole chromophores in the protein. The comparison of the quantum yield and of average lifetime showed that some tryptophan residues were completely quenched. Energy transfer from tyrosines to tryptophan residues previously detected in steady-state fluorescence was also revealed by fluorescence decay measurements. Comparison of both methods showed that an important part of transfers occurred with a very fast rate equal to or greater than 10(10) s-1. Both lifetimes and the ratio of the short and the long-lived component were found dependent on tri-N-acetylchitotriose binding.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1976.tb10845.x

Nanosecond‐Pulse Fluorimetry of Wheat‐Germ Agglutinin (Lectin)

Optimization of laser beams in FRAP experiments of microscopical objects

The fluorescence decay of S4 ribosomal protein was studied at different pH, concentrations and temperatures. A careful analysis of decay curves showed that it was possible to analyze them as a sum of two exponentials. After a thorough discussion we are led to interpret our results in terms of two protein conformations, one of which prevails under 30-S subunit reconstitution conditions.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1976.tb10386.x

Philippe Wahl

Papers

Nanosecond rotational motions of apolipoprotein C-I in solution and in complexes with dimyristoylphosphatidylcholine

Resonance energy-transfer and fluorescence intensity studies of the transport of liposome-encapsulated molecules into isolated mouse liver nuclei.

Nanosecond‐Pulse Fluorimetry of Wheat‐Germ Agglutinin (Lectin)

Optimization of laser beams in FRAP experiments of microscopical objects

Nanosecond‐Pulse Fluorometry Study of S4 Ribosomal Protein