Monitoring of in vitro and in vivo translation of green fluorescent protein and its fusion proteins by fluorescence correlation spectroscopy

BACKGROUND: Because the process of protein translation is an event of sparse molecules, the measurement requires high sensitivity. One of the candidates for studying the molecules is fluorescence correlation spectroscopy (FCS), which gleans quantitative information from fluctuating fluorescence signals in a diluted solution. METHODS: Using FCS, the translation products of expression plasmid for green fluorescent protein (GFP) and its fusion proteins were measured in vitro and in vivo. RESULTS: In in vitro translation, the number of products increased linearly for 90 min upon concentration of the plasmid. The autocorrelation function for GFP was fitted with a one-component model with a diffusion time of 0.18 ms, which was identical to the value expected from the molecular weight. In the cases of GFP- tagged hypoxia-inducible factor-1 alpha and glucocorticoid receptor, each fitting result was significantly improved with a two-component model. The slow component with a diffusion time of 6 ms appeared to be related to the ribosome or polysome. In response to the addition of dexamethasone, the nuclear translocation from cytosol clearly induced the decrease in number of molecules in the focal point. CONCLUSIONS: FCS permits monitoring of the number of molecules translated in vitro and in vivo, the translation rate, and the molecular weight.     

Use of holographic laser interferometry to study the diffusion of polymers in gels

The aim of this study was to demonstrate the potential for holographic interferometry to be used for diffusion studies of large molecules in gels. The diffusion and partitioning of BSA (67,000 g/mol) and pullulans (5,900-112,000 g/mol) in agarose gel were investigated. The gel diffusion coefficients obtained for BSA were higher when distilled water was used as a solvent compared to those obtained with 0.1 M NaCl as the solvent. Furthermore, the gel diffusion coefficient increased with increasing BSA concentration. The same trend was found for liquid BSA diffusion coefficients obtained by DLS. BSA partition coefficients obtained at different agarose gel concentrations (2-6%, w/w) decreased slightly with increasing gel concentration. However, all BSA gel diffusion coefficients measured were significantly lower than those in pure solvent and they decreased with increasing agarose concentration. The gel diffusion coefficients obtained for pullulans decreased with increasing pullulan molecular weight. The same effect from increased molecular weight was seen in the liquid diffusion coefficients measured by DLS. The pullulan partition coefficients obtained decreased with increasing molecular weight. However, pullulans with a larger Stokes' radius than BSA had partition coefficients that were higher or approximately the same as BSA. This implied that the pullulan molecules were more flexible than the BSA molecules. The results obtained for BSA in this study agreed well with other experimental studies. In addition, the magnitude of the relative standard deviation was acceptable and in the same range as for many other methods. The results thereby obtained showed that holographic interferometry is a suitable method for studying diffusion of macromolecules in gels.     

Structure of cCF10, a peptide sex pheromone which induces conjugative transfer of the Streptococcus faecalis tetracycline resistance plasmid, pCF10

The peptide pheromone, cCF10, which induces aggregation and high frequency plasmid transfer in Streptococcus faecalis cells carrying the tetracycline resistance plasmid, pCF10, was isolated and its structure determined. The molecular weight of cCF10 is 789, and its amino acid sequence is H-Leu-Val-Thr-Leu-Val-Phe-Val-OH. Pheromone activity, as determined by a clumping induction assay, was detectable at a concentration of 2.5 x 10(-11) M. A peptide of the same sequence as that of the cCF10 produced by S. faecalis cells was synthesized by the liquid-phase method. The synthetic pheromone showed biological activity and chromatographic behavior that was identical to that of the cCF10 of bacterial origin. When the response of S. faecalis cells to various concentrations of synthetic cCF10 was monitored by measuring both the frequency of plasmid transfer and the synthesis of pheromone-inducible antigens, an excellent correlation was observed between donor ability and the appearance of a 150-kilodalton protein that appears to be involved in formation of mating aggregates. The dose-response data in the range of concentrations where the amount of pheromone became limiting (10(-11)-10(-12) M) were consistent with the notion that as few as one or two molecules per donor cell may be sufficient to induce a mating response.     

Modeling of Microvascular Permeability Changes after Electroporation

Vascular endothelium selectively controls the transport of plasma contents across the blood vessel wall. The principal objective of our preliminary study was to quantify the electroporation-induced increase in permeability of blood vessel wall for macromolecules, which do not normally extravasate from blood into skin interstitium in homeostatic conditions. Our study combines mathematical modeling (by employing pharmacokinetic and finite element modeling approach) with in vivo measurements (by intravital fluorescence microscopy). Extravasation of fluorescently labeled dextran molecules of two different sizes (70 kDa and 2000 kDa) following the application of electroporation pulses was investigated in order to simulate extravasation of therapeutic macromolecules with molecular weights comparable to molecular weight of particles such as antibodies and plasmid DNA. The increase in blood vessel permeability due to electroporation and corresponding transvascular transport was quantified by calculating the apparent diffusion coefficients for skin microvessel wall (D [μm²/s]) for both molecular sizes. The calculated apparent diffusion coefficients were D = 0.0086 μm²/s and D = 0.0045 μm²/s for 70 kDa and 2000 kDa dextran molecules, respectively. The results of our preliminary study have important implications in development of realistic mathematical models for prediction of extravasation and delivery of large therapeutic molecules to target tissues by means of electroporation.     

Physical properties and gel electrophoresis behavior of R12-derived plasmid DNAs.

A series of closed circular (I) plasmid DNAs has been derived from drug resistance factor R12, and the nicked circular (II) and linear (III) derivatives of these molecules prepared by irradiation in the presence of ethidium bromide and by treatment with restriction enzyme EcoRI, respectively. These DNAs encompass the molecular weight range 3.6 to 61 megadaltons. The base compositions range from 45% to 51% (GC) as estimated by buoyant density determinations. The smaller plasmids are significantly less supercoiled (9-10%) than are the larger (12-13%). The gel electrophoretic behavior of the three DNA structural forms was determined as a function of molecular weight in agarose gels of concentrations ranging from 0.7% to 1.6% and at electrophoresis salt concentrations from 0.02 M to 0.08 M sodium acetate. The mobilities of DNAs I and III undergo a reversal relative to each other at a molecular weight which decreases with increasing agarose gel concentration. The molecular weight at which DNA II fails to enter a gel depends upon the ionic strength during electrophoresis but not upon the gel concentration.     

Bacteriophage phiNS11: a lipid-containing phage of acidophilic thermophilic bacteria. IV. Sedimentation coefficient, diffusion coefficient, partial specific volume, and particle weight of the phage.

The particle weight (molecular weight) of phiNS11 was determined from the sedimentation coefficient, diffusion coefficient, and partial specific volume of the phage. The sedimentation coefficient of the phage (S(0)20, W) is 416 +/- 2.7S. The diffusion coefficient D(0)20, W), which was determined by quasielastic light scattering measurement, is (0.57 +/- 0.03) x 10(-7) cm2/s. The partial specific volume was determined by the mechanical oscillation technique to be 0.747 +/- 0.007 cm3/g. Based on these values, the particle weight of the phage was calculated to be (70.3 +/- 4.3) x 10(6) daltons, which agrees well with the particle weight (69--72 x 10(6) daltons) estimated from the molecular weight of phage DNA and the content of DNA. The Stokes radius of the phage particle was calculated to be 37.7 +/- 2 nm and hydration of the phage was estimated to be 1.18 cm3/g of dry phage. From the particle weight and the chemical composition of the phage, we estimated that one phage particle contains one double-stranded DNA molecule, 16,000 residues of fatty acid, 72 protein I molecules, 920 protein II, 42 protein III, 48 protein IV, 290 protein V molecules, and 3,700 molecules of polyamines.     

Unified description of electrophoresis and diffusion for DNA and other polyions

The electrophoretic mobilities and diffusion coefficients of single- and double-stranded DNA molecules up to 50,000 bases or base pairs in size have been analyzed, using mobilities and diffusion coefficients either measured by capillary electrophoresis or taken from the literature. The Einstein equation suggests that the electrophoretic mobilities (mu) and diffusion coefficients (D) should be related by the expression mu/D = Q/k(B)T, where Q is the charge of the polyion (Q = ze(o), where z is the number of charged residues and e(o) is the fundamental electronic charge), k(B) is Boltzmann's constant, and T is the absolute temperature. If this equation were true, the ratio mu/zD should be a constant equal to e(o)/k(B)T (39.6 V(-1)) at 20 degrees C. However, the ratio mu/zD decreases with an increase in molecular weight for both single- and double-stranded DNAs. The mobilities and diffusion coefficients are better described by the modified Einstein equation mu/N(m)D = e(o)/k(B)T, where N is the number of repeat units (bases or base pairs) in the DNA and m is a constant equal to the power law dependence of the diffusion coefficients on molecular weight. The average value of the ratio mu/N(m)D is 40 +/- 4 V(-1) for 36 single- and double-stranded DNA molecules of different sizes, close to the theoretically expected value. The generality of the modified Einstein equation is demonstrated by analyzing literature values for sodium polystyrenesulfonate (PSS). The average value of the ratio mu/N(m)D is 35 +/- 6 V(-1) for 14 PSS samples containing up to 855 monomers.     

Determination of diffusion coefficients in biofilms by confocal laser microscopy

Microbial exopolymer may hinder the diffusion of nutrients, antibiotics, and other materials to the cell surface. Studies of diffusion in biofilms have been limited to indirect measurements. This study demonstrated the use of fluorescein and size-fractionated fluor-conjugated dextrans in conjunction with scanning confocal laser microscopy to directly monitor and determine diffusion coefficients within biofilms. The monitoring approaches were simple and, when combined with computerized image collection, allowed assembly of a data set suitable for calculation of one-dimensional diffusion coefficients for biofilm regions. With these techniques, it was shown that regional variability in the mobility of the dextrans occurred within mixed-species biofilms. Some regions exhibited rapid diffusion of all test molecules, while adjacent regions were only penetrated by the lower-molecular-weight compounds. The effective diffusion coefficients (D(e)) determined in a mixed-species biofilm were a function of the molecular radius of the probe (i.e., fluorescein, D(e) = 7.7 x 10 cm s; 4,000 molecular weight, D(e) = 3.1 x 10 cm s; and 2,000,000 molecular weight, D(e) = 0.7 x 10 cm s). These results demonstrated that diffusion in the biofilm was hindered relative to diffusion in the bulk solution. The study indicated that in situ monitoring by scanning laser microscopy is a useful approach for determining the mobility of fluorescently labeled molecules in biofilms, allowing image acquisition, appropriate scales of study, both xy and xz monitoring, and calculation of D(e) values.     

Characterization of Phospholipid Mixed Micelles by Translational Diffusion

The concentration dependence of the translational self diffusion rate, D (s), has been measured for a range of micelle and mixed micelle systems. Use of bipolar gradient pulse pairs in the longitudinal eddy current delay experiment minimizes NOE attenuation and is found critical for optimizing sensitivity of the translational diffusion measurement of macromolecules and aggregates. For low volume fractions Phi (Phi\\ le 15% v/v) of the micelles, experimental measurement of the concentration dependence, combined with use of the D (s)= D (o)(1-3.2lambdaPhi) relationship, yields the hydrodynamic volume. For proteins, the hydrodynamic volume, derived from D (s) at infinitely dilute concentration, is found to be about 2.6 times the unhydrated molecular volume. Using the data collected for hen egg white lysozyme as a reference, diffusion data for dihexanoyl phosphatidylcholine (DHPC) micelles indicate approximately 27 molecules per micelle, and a critical micelle concentration of 14 mM. Differences in translational diffusion rates for detergent and long chain phospholipids in mixed micelles are attributed to rapid exchange between free and micelle-bound detergent. This difference permits determination of the free detergent concentration, which, for a high detergent to long chain phospholipid molar ratio, is found to depend strongly on this ratio. The hydrodynamic volume of DHPC/POPC bicelles, loaded with an M2 channel peptide homolog, derived from translational diffusion, predicts a rotational correlation time that slightly exceeds the value obtained from peptide (15)N relaxation data.     

The effect of spermine on plasmid condensation and dye release observed by fluorescence correlation spectroscopy

We demonstrate that fluorescence correlation spectroscopy (FCS) can be employed to follow the conformational changes of DNA molecules induced by the addition of a cationic condensing compound (spermine). In our experiments the plasmid pHbetaAPr-1-neo (10 kbp; contour length 3.4 microm) was labeled with propidium iodide (PrIo) and then titrated with spermine to induce its condensation. When spermine was applied at concentrations above 5 microM (spermine/DNAphosphate=0.375), the diffusion time of the labeled plasmid dropped from 15 ms down to 3 ms (its diffusion coefficient, D, increased from 1.0x10(-12) m2/s to 6.0x10(-12) m2/s). The application of spermine was also accompanied by decreasing count rate and particle number, reflecting the dye's dissociation. The data presented show that FCS may become a valuable tool in studying supramolecular aggregate formation, especially when association is followed by a change in the hydrodynamic size of the resulting complex.     

Estimation of diffusion coefficients of proteins

A correlation for estimating the diffusion coefficients of protein molecules is presented. The correlation is based upon literature values of the protein diffusion coefficients and molal volumes for 143 proteins. The correlation can be used for the estimation of diffusion coefficients using only molecular weight. Accuracy is such that a linear regression on 301 proteins showed 75% of the diffusion coefficients estimated fell within 20% of the experimental values. The relationship between this correlation, the Stokes–Einstein equation, and the Wilke–Chang correlation is discussed.     

Study of the translational diffusion of macromolecules in beads of gel chromatography by the FRAP method

We measured the translational diffusion of fractions of dextrans labelled with fluorescein isothiocyanate, in Sephadex gel beads permeated by aqueous solutions of these molecules. The molecular weights of these fractions were between 5400 and 200,000 and measurements of their diffusion coefficients inside a gel bead (D) and in the free solution (D0), were performed using the fluorescence recovery after photobleaching method (FRAP). We also determined the coefficient of partitioning (Kav) of these fractions between the gel and the free solvent, with a new microfluorimetric method. We found that, for Sephadex G-50, G-75, G-100, G-150 and G-200 gels, Kav varied with the Stokes radius (rs) of the dextran molecules, in agreement with the formula of Laurent and Killander (J. Chromatogr. 14 (1964) 317). For Sephadex G-100, G-150 and G-200 gels, D/D0 varied with rs, according to the theory of Ogston et al. (Proc. R. Soc. Lond. 333 (1973) 297). In addition, these theories predict a relation linking D/D0 to Kav which was well verified. Our work is the first systematic study of the translational diffusion of macromolecules in a chromatography gel. These measurements should allow a better evaluation of the factors which influence the resolution in exclusion chromatography. In addition, the diffusion of macromolecules in gels may provide models for the diffusion of these molecules in the cytoplasm of living cells and in connective biological tissues.     

Plasmid deoxyribonucleic acid in clinical isolates of Neisseria gonorrhoeae.

Two different sizes of circular covalently closed deoxyribonucleic acid plasmids have been identified in four independent clinical isolates of eisseria gonorrhoeae. All four strains contained a small plasmid with a molecular weight of 2.8 X 10-6 and two of the four stains also contained a large plasmid with a molecular weight of 24.5 X 10-6. The avirulent derivative of each of these four strains had the same plasmid complement as its virulent parent. There was no correlation between the presence of these plasmids and antibiotic resistance, piliation, and colony type associated with virulence, or ability to grow without seven specific amino acid supplements.     

A novel correlation for protein diffusion coefficients based on molecular weight and radius of gyration

A new correlation is proposed for the prediction of protein diffusion coefficients in free solution. Molecular weight and radius of gyration of proteins are employed as correlation parameters in this method. Both parameters can be easily found in the literature. The correlation works well for diverse proteins with different shapes and extensive molecular weight. Furthermore, this method does not require a preassumption regarding the protein shape while it offers a rapid and convenient calculation with a high accuracy. Also, the proposed correlation can elucidate the estimation deviation of previous correlation methods in the literature.     

Comparison of molecular weight determination of sodium alginate by sedimentation-diffusion and light scattering

The weight average molecular weight, M̄_w, of sodium alginates were determined by the sedimentation-diffusion technique using photon correlation spectroscopy rather than boundary spreading in the analytical ultracentrifuge to determine the translational diffusion coefficients. This enables the diffusion coefficients to be determined easily and accurately. Excellent correlation is found between the observed zero concentration translational diffusion coefficient, D_O and the M̄_W values in a emphirical power law. The M̄_W obtained by sedimentation-diffusion and laser light scattering compare very favourably. The concentration dependence of the photon correlation spectroscopy data allowed determination of the coil overlap concentration, c*. The inverse proportionality of c* to both M̄_W and [η] is demonstrated.     

Conjugal Transfer of Plasmid-Borne Multiple Antibiotic Resistance in Streptococcus faecalis var. zymogenes

A strain of Streptococcus faecalis var. zymogenes, designated JH1, had high-level resistance to the antibiotics streptomycin, kanamycin, neomycin, erythromycin, and tetracycline. These resistances were lost en bloc from approximately 0.1% of cells grown in nutrient broth at 45 C. The frequency of resistance loss was not increased by growth in the presence of the "curing" agents acriflavine or acridine orange, but after prolonged storage in nutrient agar 17% of cells became antibiotic sensitive. Covalently closed circular deoxyribonucleic acid (DNA) molecules were isolated from the parental strain and from antibiotic-sensitive segregants by using cesium chloride-ethidium bromide gradients. DNA molecular species were identified by using neutral sucrose gradients. Strain JH1 contained two covalently closed circular DNA species of molecular weights 50 x 10(6) and 38 x 10(6). An antibiotic-sensitive segregant, strain JH1-9, had lost the larger molecular species. A second sensitive segregant, strain JH1-5, had also lost the larger molecular species but a new molecular species of approximate molecular weight 6 x 10(6) was present. The antibiotic resistances that were curable from the parental strain were transferred to antibiotic-sensitive strains of S. faecalis and to strain JH1-9, during mixed incubation in nutrient broth at 37 C. Data to be described are interpreted to suggest that the transfer is by a conjugal mechanism. Analysis of the plasmid species in recipient clones showed that all had received the plasmid of molecular weight 50 x 10(6). Strain JH1-5 was not a good recipient. Analysis of one successful recipient clone of JH1-5 revealed that it had gained the 50 x 10(6) molecular weight plasmid but lost the 6 x 10(6) molecular weight species. These data are interpreted to mean that the multiple antibiotic resistance is borne by a transferable plasmid of 50 x 10(6) molecular weight, and that in clone JH1-5 this plasmid suffered a large deletion leaving only a 6 x 10(6) remnant which was incompatible with the complete replicon.     

Diffusion of fluorescently labeled macromolecules in cultured muscle cells.

Myotubes were obtained from culture of satellite cells. They had a sarcomeric organization similar to that of muscle. The diffusion in the direction perpendicular to the fibers of microinjected fluorescein isothiocyanate-dextrans of molecular weight ranging from 9500 to 150,000 was examined by modulated fringe pattern photobleaching. On the time scale of the observation, 10-30 S, all of the dextrans were completely mobile in the cytoplasm. The diffusion coefficients were compared to the values obtained in water. The ratio D(cytoplasm)/D(w) decreased with the hydrodynamic radius R(h) of the macromolecules. The mobility of inert molecules in muscle cells is hindered by both the crowding of the fluid phase of the cytoplasm and the screening effect due to myofilaments: D(cytoplasm)/D(w) = (D/D(w)) protein crowding x (D/D(w))(filament screening). The equation (D/D(w))filament screening = exp(-K(L)RCh) was used for the contribution of the filaments to the restriction of diffusion. A free protein concentration of 135 mg/ml, a solvent viscosity of cytoplasm near that of bulk water, and a calculated K(L) of 0.066 nm(-1), which takes into account the sarcomeric organization of filaments, accurately represent our data.     

Dependence of antithrombin III and thrombin binding stoichiometries and catalytic activity on the molecular weight of affinity-purified heparin

Heparin was fractionated by affinity chromatography on immobilized antithrombin III followed by gel filtration on Sephadex G-100. Eighteen fractions were obtained ranging in molecular weight from 9,700 to 34,300 as determined by sedimentation equilibrium. The binding stoichiometries of antithrombin III and thrombin interactions with the heparin of these fractions were measured, using changes in intrinsic and extrinsic fluorescence. Catalytic activity also was measured for each of the heparin fractions. As the molecular weight of heparin varied from about 10,000 to 30,000, the average number of antithrombin and thrombin sites/heparin molecule varied from 1.0 to 2.1 and 2.4 to 6.8. In addition, the molar specific activity increased 5.7-fold, an increase which correlated directly with the product of the number of antithrombin III and thrombin molecules bound. Thus as the number of bound molecules increased with increased molecular weight, the rate of reaction/bound antithrombin III increased in proportion to the number of bound thrombin molecules and vice versa. This can be explained by assuming that heparin functions as a template for both proteins, that all bound thrombin and antithrombin III molecules are accessible to each other, and that the rate at which a bound molecule reacts is proportional to the number of molecules of its interacting counterpart bound. These observations and conclusions are similar to those of Hoylaerts et al. (Hoylaerts, M., Owen, W. G., and Collen, D. (1984) J. Biol. Chem. 259, 5670-5677), who demonstrated that the rate at which single molecules of antithrombin III, covalently attached to heparin, react increases as the thrombin binding capacity (chain length) of heparin increases.     

Evidence for a nonrandom base sequence in a Bacillus pumilus plasmid: EcoR1 endonuclease digestion of pPL576.

EcoR1 endonuclease digested the Bacillus pumilus plasmid pPL576 (molecular weight similar to 28 X 10-6) into three distinct size classes of linear fragments. The molecular weights of the fragments are 13.0 X 10-6, 0.5 X 10-6, and 6.5 X 10-6 by sucrose gradient analysis. By electron microscope analysis the three fragments account for about 99% of the intact plasmid. These results indicate that pPL576 molecules contain a nonrandom base sequence, and are consistent with the interpretation that pPL576 is autonomous and not the result of cyclization of random chromosome fragments.