Showing papers in "Biochemistry in 1981"

Thermodynamics of protein association reactions: forces contributing to stability

Abstract: Reviewing the thermodynamic parameters characterizing self-association and ligand binding of proteins at 25 OC, we find AGO, AHo, AS\", and ACpo are often all of negative sign. It is thus not possible to account for the stability of association complexes of proteins on the basis of hydrophobic interactions alone. We present a conceptual model of protein association consisting of two steps: the mutual penetration of hydration layers causing disordering of the solvent followed by further short-range interactions. The net AGO for the complete association process is primarily determined by the positive entropy change accompanying the first step and the negative enthalpy change of the second step. On the basis of the thermochemical behavior of small molecule interactions, we conclude that the strengthening of hydrogen bonds in the I n the past decade, a complete thermodynamic description of the self-association of many proteins and their interactions From the Laboratory of Molecular Biology (P.D.R.) and Laboratory of Nutrition and Endocrinology (S.S.), National Institute of Arthritis, Metabolism and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20205. Received September 23, 1980. low dielectric macromolecular interior and van der Waals' interactions introduced as a consequence of the hydrophobic effect are the most important factors contributing to the observed negative values of AHo and ASo and hence to the stability of protein association complexes. The X-ray crystallographic structures of these complexes are consonant with this analysis. The tendency for protein association reactions to become entropy dominated and/or entropy-enthalpy assisted at low temperatures and enthalpy dominated at high temperatures (a consequence of the typically negative values of AC,\") arises from the diminution of the hydrophobic effect with increasing temperature which is a general property of the solvent, water. with small molecular substrates has become available. Concomitantly, X-ray crystallography has provided a detailed picture of some of these associations, and this has stimulated a number of theoretical studies (Levitt & Warshel, 1975; Gelin & Karplus, 1975; Chothia & Janin, 1975), based upon energetic considerations, to account for these structures. The This article not subject to U S . Copyright. Published 1981 by the American Chemical Society T H E R M O D Y N A M I C S O F P R O T E I N A S S O C I A T I O N V O L . 2 0 , N O . 1 1 , 1 9 8 1 3097 Table I: Thermodynamics of Protein Association' association process A G \" ~ AiY A s o , A c p o (kcal mol-') (kcal mol-l) (cal K-I mol-') (cal K-I mol-I) refb trypsin (bovine) + inhibitor (soybean) -14.6 8.6 78 -440 c, d deoxyhemoglobin S gelation -3.4 2.0 18 -200 e, f lysozyme self-association (indefinite) -3.9 -6 .4 -8.3 g glucagon trimerization -12.1 -3 1 -64 -430 h, i hemoglobin t haptoglobin -11.5 -3 3 -7 3 -940 i a-chymotrypsin dimerization -7.1 -35 -9 5 k, I S-peptide + S-protein (ribonuclease) -13 -40 -90 -1100 m, n All thermodynamic parameters expressed per mole of complex formed except the indefinite association cases of hemoglobin S and lysozyme for which the mole refers to the monomeric protein reacted. Unitary entropy and free energy are given for processes of defined stoichiometry. Standard states are hypothetical 1 M protein, pH at which the reaction was measured. All pHs were close to 7 except for trypsin, pH 5, haptoglobin, pH 5.5, and glucagon, pH 10.5. All data for 25 \"C except glucagon, T = 30 \"C. ence is to calorimetric work and the second is to X-ray crystallographic structure determination. al. (1974). e Rosset al. (1977). Wishner e t al. (1975). g Banerjee et al. (1975). Johnson et al. (1979). * Sasaki et al. (1975). For each entry, the first referSweet et Baugh & Trowbridge (1972). Lavialle et al. (1974). Shiao & Sturtevant (1969). lVandlen &Tulinsky (1973). Hearn et al. (1971). Wyckoff e t al. (1970). methodology and problems involved in such calculations have been critically reviewed by NBmethy & Scheraga (1977). In this paper we review the thermodynamics of protein association processes for the examples best characterized in terms of their chemistry and structure. From this survey we find that the thermodynamic parameters AGO, Ai?, AS\", and ACpo are predominantly of negative sign. This result poses severe difficulties for interpretations of protein association based upon the entropically driven hydrophobic effect. The aim of this paper is to attempt to account for the signs and magnitudes of these thermodynamic parameters for protein association reactions in terms of known molecular forces and the thermochemistry of small molecule interactions.

Estimation of globular protein secondary structure from circular dichroism.

Abstract: A new method is developed in which a circular dichroism (CD) spectrum is analyzed directly as a linear combination of the CD spectra (from 190 to 240 nm) of 16 proteins whose secondary structures are known from X-ray crystallography. This avoids the dilemma encountered in previous methods of trying to define single reference CD spectra that were supposed to characterize such broad and variable classes as helix, beta sheet, beta turn, and "remainder". It also permits a more accurate and flexible analysis. The usual instability in using so many parameters is automatically controlled by a simple constrained statistical regularization procedure (similar to ridge regression). Sixteen tests were made by removing 1 spectrum at a time from the set of 16 and analyzing it in terms of the other 15. The product moment correlation coefficients between the computed fractions of helix, beta sheet, beta turn, and remainder and the fractions from the X-ray data were 0.96, 0.94, 0.31,, and 0.49, respectively. Thus, the helix and beta-sheet accuracy is very good. (The corresponding values calculated by a previous method with four reference spectra were 0.85, 0.25, --0.31, and 0.46.).

Crystallographic refinement and atomic models of a human Fc fragment and its complex with fragment B of protein A from Staphylococcus aureus at 2.9- and 2.8-A resolution.

Abstract: The model of human Fc fragment was refined at 2.9-A resolution. Two different automated procedures for crystallographic refinement were used [Deisenhofer, J., & Steigemann, W. (1975) Acta Crystallogr., Sec. B B31, 238; Jack, A., & Levitt, M. (1978) Acta Crystallogr., Sect. A A34, 931]. The final R value is 0.22. The dimer of CH3 domains closely resembles the CH1-CL aggregate in Fab fragments. There is no contact between CH2 domains. The contact between CH2 and CH3 domains has about one-third of the size of the CH3-CH3 contact. The carbohydrate, a branched chain of nine hexose units, covers parts of the C-contact face of the CH2 domain, shielding hydrophobic residues on this surface. Six atoms of the carbohydrate are within hydrogen-bonding distance of atoms in the CH2 domain. Crystallographic refinement of the complex between Fc fragment and fragment B of protein A from Staphylococcus aureus reduced the R value of the model is 0.24. A major part of the structure of fragment B consists of two alpha helics; the rest of the polypeptide chain is folded irregularly. In the crystal, fragment B forms two contacts with Fc fragment molecules. Contact 1 involves residues from both helices of fragment B, and residues from the CH2 and CH3 domains of FC, and is predominantly hydrophobic. Contact 2 is smaller than contact 1. Residues from the second helix and adjacent residues of fragment B and residues only from the CH3 domain of Fc contribute to contact 2. The nature of contact 2 is mainly polar and includes a sulfate ion. There are strong arguments that contact 1 is the fragment B-Fc contact formed in solution under physiological conditions, while contact 2 is a crystal contact.

Use of resonance energy-transfer to monitor membrane-fusion

Abstract: An assay for vesicle--vesicle fusion involving resonance energy transfer between N-(7-nitro-2,1,3-benzoxadiazol-4-yl), the energy donor, and rhodamine, the energy acceptor, has been developed. The two fluorophores are coupled to the free amino group of phosphatidylethanolamine to provide analogues which can be incorporated into a lipid vesicle bilayer. When both fluorescent lipids are in phosphatidylserine vesicles at appropriate surface densities (ratio of fluorescent lipid to total lipid), efficient energy transfer is observed. When such vesicles are fused with a population of pure phosphatidylserine vesicles by the addition of calcium, the two probes mix with the other lipids present to form a new membrane. This mixing reduces the surface density of the energy acceptor resulting in a decreased efficiency of resonance energy transfer which is measured experimentally. These changes in transfer efficiency allow kinetic and quantitative measurements of the fusion process. Using this system, we have studied the ability of phosphatidylcholine, phosphatidylserine, and phosphatidylcholine--phosphatidylserine (1:1) vesicles to fuse with cultured fibroblasts. Under the conditions employed, the majority of the cellular uptake of vesicle lipid could be attributed to the adsorption of intact vesicles to the cell surface regardless of the composition of the vesicle bilayer.

Diffusion-Driven Mechanisms of Protein Translocation on Nucleic Acids. 1. Models and Theory?

Abstract: Genome regulatory proteins (e.g., repressors or polymerases) that function by binding to specific chromosomal target base pair sequences (e.g., operators or promoters) can appear to arrive at their targets at faster than diffusion-controlled rates. These proteins also exhibit appreciable affinity for nonspecific DNA, and thus this apparently facilitated binding rate must be interpreted in terms of a two-step binding mechanism. The first step involves free diffusion to any nonspecific binding site on the DNA, and the second step comprises a series of protein translocation events that are also driven by thermal fluctuations. Because of nonspecific binding, the search process in the second step is of reduced dimensionality (or volume); this results in an accelerated apparent rate of target location. In this paper we define four types of processes that may be involved in these protein translocation events between DNA sites. These are (i) "macroscopic" dissociation--reassociation processes within the domain of the DNA molecule, (ii) "microscopic" dissociation--reassociation events between closely spaced sites in the DNA molecule, (iii) "intersegment transfer" (via "ring-closure") processes between different segments of the DNA molecule, and (iv) "sliding" along the DNA molecule. We present mathematical and physical descriptions of each of these processes, and the consequences of each for the overall rate of target location are worked out as a function of both the nonspecific binding affinity between protein and DNA and the length of the DNA molecule containing the target sequence. The theory is developed in terms of the Escherichia coli lac repressor--operator interaction since data for testing these approaches are available for this system [Barkley, M. (1981) Biochemistry 20, 3833; Winter, R. B., & von Hippel, P. H. (1981) Biochemistry (second paper of three in this issue); Winter, R. B., Berg, O. G., & von Hippel, P. H. (1981) Biochemistry (third paper of three in this issue)]. However, we emphasize that this approach is general for the analysis of mechanisms of biological target location involving facilitated transfer processes via nonspecific binding to the general system of which the target forms a small part.

Mechanism of protein stabilization by glycerol: preferential hydration in glycerol-water mixtures

Abstract: A densimetric investigation of the interactions between solvent components in glycerol-water mixtures (between 10 and 40 vol % glycerol) and seven proteins have been carried out in the acid pH region. All the proteins were found to be preferentially hydrated at all conditions used, i.e., addition of the proteins to the mixed solvent results in an increase in the chemical potential of glycerol. It is considered that this thermodynamically unfavorable interaction should tend to minimize the surface of contact between proteins and glycerol and in this way stabilize the native structure of globular proteins.

Structural identification of autoinducer of Photobacterium fischeri luciferase.

Abstract: Synthesis of bacterial luciferase in some strains of luminous bacteria requires a threshold concentration of an autoinducer synthesized by the bacteria and excreted into the medium. Autoinducer excreted by Photobacterium fischeri strain MJ-1 was isolated from the cell-free medium by extraction with ethyl acetate, evaporation of solvent, workup with ethanol-water mixtures, and silica gel chromatography, followed by normal-phase and then reverse-phase high-performance liquid chromatography. The final product was greater than 99% pure. The structure of the autoinducer as determined by high-resolution 1H nuclear magnetic resonance spectroscopy, infrared spectroscopy, and high-resolution mass spectrometry was N-(3-oxohexanoly)-3-aminodihydro-2(3H)-furanone [or N-(beta-ketocaproyl)homoserine lactone]. The formation of homoserine by hydrolysis of the autoinducer was consistent with this structure. Synthetic autoinducer, obtained as a racemate, was prepared by coupling homoserine lactone to the ethylene glycol ketal of sodium 3-oxohexanoate, followed by mildly acidic removal of the protecting group; this synthetic material showed the appropriate biological activity.

Affinities of Amino Acid Side Chains for Solvent Water

Abstract: Equilibria of distribution of amino acid side chains, between their dilute aqueous solutions and the vapor phase at 25 degrees C, have been determined by dynamic vapor pressure measurements. After correction to pH 7, the resulting scale of "hydration potentials", or free energies of transfer from the vapor phase to neutral aqueous solution, spans a range of approximately 22 kcal/mol. The side chain of arginine is much more hydrophilic than those of the other common amino acids, with an equilibrium constant of approximately 10(15) for transfer from the vapor phase to neutral aqueous solution. Hydration potentials are more closely correlated with the relative tendencies of the various amino acids to appear at the surface of globular proteins than had been evident from earlier distribution studies on the free amino acids. Both properties are associated with a pronounced bias in the genetic code.

Phospholipid vesicle formation and transmembrane protein incorporation using octyl glucoside

Abstract: Removal of detergent from mixed micelles of egg yolk phosphatidylcholine and octyl glucoside leads to formation of unilamellar phospholipid vesicles with a diameter of about 230 nm. The same procedure applied to mixed micelles containing the transmembrane protein glycophorin A, in addition to lipid and detergent, produces vesicles of the same size with glycophorin incorporated into the bilayer. The pure lipid vesicles are highly impermeable to both anions and cations, and incorporation of up to 220 molecules of glycophorin per vesicle has little effect on permeability.

Diffusion-driven mechanisms of protein translocation on nucleic acids. 2. The Escherichia coli repressor--operator interaction: equilibrium measurements.

Abstract: In this paper the equilibrium binding of lac re- pressor to operator sites has been studied as a function of monovalent salt concentration, of length of the DNA molecule containing the operator, and (by using various natural lac “pseudo”-operators) of operator base pair sequence. The nitrocellulose filter assay has been used to obtain values of repressor-operator association constants (KRO), both directly and as ratios of association to dissociation rate constants (k,/kd). Measurements of KRo have been made in the absence of MgZf or other divalent ions, allowing a direct estimate [Record, M. T., Jr., Lohman, T. M., & deHaseth, P. L. (1976) J. Mol. Biol. 107, 1451 of the contribution of electrostatic (charge-charge) interactions to the stability of the RO com- plexes. Using lac operator containing DNA restriction frag- ments of known size, we have shown the following: (i) The magnitide of the RO interaction is salt concentration de- pendent. A plot of log

Information content in the circular dichroism of proteins.

Abstract: A method is presented for predicting the secondary structure of a protein from its circular dichroism (CD) spectrum. Eight types of secondary structures are considered: helix; parallel and antiparallel beta strand; types I, II, and III beta turn; all other beta turns combined; and "other" structures. The method is based on mathematical calculation of orthogonal basis CD spectra from the CD spectra of proteins with known secondary structure. Five basis CD spectra are needed to reconstruct the 16 original protein CD spectra that extend into the vacuum ultraviolet region to 178 nm. Thus, one can expect to extract five independent pieces of information from the CD spectrum of a protein. Each basis CD spectrum corresponds to a known mixture of secondary structures so that the coefficients that reconstruct the protein CD spectrum can also be used to predict secondary structure. Furthermore, when the same method is applied to protein secondary structure rather than CD, it is found that only five basis secondary structure vectors are needed to reconstruct the original protein secondary structure vectors. Thus there are five independent "superstructures", consisting of a mixture of standard secondary structures, in the proteins studied. It would appear that there is enough information in the CD spectrum of a protein to predict all types of secondary structure. Our CD analyses compare favorably with the X-ray data.

Diffusion-driven mechanisms of protein translocation on nucleic acids. 3. The Escherichia coli lac repressor-operator interaction: kinetic measurements and conclusions

Abstract: The association and dissociation kinetics of the Escherichia coli lac repressor--operator (RO) complex have been examined as a function of monovalent ion concentration and operator-containing DNA fragment length in order to investigate the mechanisms used by repressor in locating (and dissociating from) the operator site. Association rate constants (ka) measured with an 80- or a 203-base-pair lac operator containing DNA fragment are 3--5-fold smaller than those determined with a 6700-base-pair operator fragment or with intact lambda plac5 DNA (50000 base pairs) at all salt concentrations tested. At salt concentrations less than approximately 0.1 M KCl, association rate constants to all operator-containing DNA fragments (except lambda plac5 DNA) are insensitive to variations in salt concentration, but the limiting low salt value of ka appears to depend upon operator-containing DNA length. The value of ka for lambda plac5 DNA decreases significantly from the approximately 0.1 M KCl maximum at low salt. Above approximately 0.1 M KCl, repressor--operator association rate constants for all operator-containing DNA substrates tested show a similar decrease with increasing salt concentration, which does not appear to depend upon the length of the DNA molecule (except for the very small DNA fragments). In contrast to the association reaction, kd, the dissociation rate constant, decreases linearly (on a log kd vs. log [KCl] plot) with decreasing salt concentration over virtually the entire salt concentration range studied (0.05--0.2 M KCl). These results are consistent with the explanation of the unusually fast association kinetics for this system in terms of a two-step model in which repressor initially diffuses to a nonoperator DNA binding site (forming an RD complex) and then rapidly "scans" (in a locally correlated fashion) adjacent sites until the operator is located or the repressor dissociates from the chain. Dissociation of the RO complex follows the same two-step process in reverse. Quantitative comparisons are made between these results and the theoretical predictions of the two facilitating translocation mechanisms (one-dimensional "sliding" along the DNA double helix and direct transfer between DNA segments) developed in the first paper of this series [Berg, O. G., Winter, R. B., & von Hippel, P. H. (1981) Biochemistry (first paper of three in this issue)]. We conclude that the experimental data for the "faster-than-diffusion-controlled" interaction of repressor and operator can be quantitatively modeled by a two-step process in which sliding is the dominant transfer mechanism. Molecular models of the initial nonspecific binding event (including "hopping") as well as sliding and interchain transfer are discussed, and the possible roles of facilitated translocation mechanisms of the diffusion-driven type in this and other in vitro and in vivo protein--nucleic acid interaction processes are considered.

1-[4-(Trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene: synthesis, fluorescence properties, and use as a fluorescence probe of lipid bilayers.

Abstract: 1-[4-(Trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH), a cationic analogue of diphenylhexatriene (DPH), has photophysical properties that are generally similar to those of DPH. In solution the fluorescence lifetime (tau) of TMA-DPH is short (less than 1.5 ns), but tau increases to approximately 7 ns when the probe is embedded in lipid bilayers at temperatures less than the thermal transition temperature (Tc) of the lipid. The cationic charge ensures that the probe is anchored at the lipid-water interface, most likely with the DPH moiety intercalated between the upper portions of the fatty acyl chains. The profiles of changes in steady-state anisotropies (rss) and limiting hindered anisotropies (r infinity) are similar for both TMA-DPH and DPH embedded in lipid bilayers, but r infinity values for TMA-DPH even at T much greater than Tc are generally greater than 0.14, e.g., at 35 degrees C in 1,2-dimyristoylglycero-3-phosphocholine (DMPC) (cf. 0.03 for DPH in DMPC at 35 degrees C). Electrostatic interactions of the cationic probe with head groups of phospholipids do not appear to significantly influence the apparent dynamics of the probe. TMA-DPH should prove useful in the study of the dynamics of phospholipid monolayers, e.g., in native or reconstituted lipoproteins.

Thermodynamic and kinetic examination of protein stabilization by glycerol

Abstract: The effect of concentrated glycerol on the thermal transitions of chymotrypsinogen and ribonuclease has been examined by differential spectrophotometry at 293 and 287 mm, respectively. It was found that for both proteins addition of glycerol raises the transition temperature, the increase in Tm being greater for ribonuclease than for chymotrypsinogen. This increase in the free energy of denaturation appears to reflect primarily a decrease in the entropy change. Analysis in terms of the Wyman linkage equation shows that, for both proteins, the exclusion of glycerol from the protein domain increases on denaturation i.e., the chemical potential of glycerol becomes even more positive when the protein unfolds relative to the native structure. This provides the thermodynamic stabilization free energy. Results of the kinetic examination of the slow unfolding reaction are consistent with the concept that the preferential exclusion of glycerol is related, at least in part, to enhanced solvent ordering.

Developmental biochemistry of cottonseed embryogenesis and germination: changing messenger ribonucleic acid populations as shown by in vitro and in vivo protein synthesis.

Abstract: Changes in messenger ribonucleic acid (mRNA) populations during embryogenesis of cottonseed have been followed by cataloging (a) extant proteins, (b) proteins synthesized in vivo, and (c) proteins synthesized in vitro from extracted RNA, all at specific stages of embryogenesis. Evidence is presented for the existence of five mRNA subsets, all apparently under different regulatory regimes, that produce the abundant proteins of embryogenesis. One of these functions principally during the cell division phase of embryogenesis and encodes among its products the seed storage proteins whose mRNA is superabundant during this period. This subset has disappeared from the abundant group by the mature seed stage. Two other subsets appear in late embryogenesis, one of which may result from the removal of the embryo from the maternal environment, since it is inducible by excision of the young embryo from the seed. The other appears to be induced by the plant growth regulator abscisic acid, whose endogenous concentration increases at this stage. It can be induced by incubating excised young embryos in abscisic acid. The last two subsets exist throughout embryogenesis, but only one of them appears to function in germination.

Enhancement of calcium uptake and phosphatidylinositol turnover by epidermal growth factor in A-431 cells.

Abstract: Epidermal growth factor (EGF) stimulates the incorporation of 32Pi and [3H]inositol into phosphatidylinositol (5-10-fold) in A-431 cells. EGF also stimulates the incorporation of 32Pi into phosphatidic acid (up to 10-fold). These effects are attributed to an acceleration of the turnover of phosphatidylinositol as a consequence of the binding of EGF to its membrane receptor. The extent of the phosphatidylinositol response to EGF parallels the extent of hormone binding. The phosphatidylinositol response to EGF appears to be dependent on an influx of calcium since (a) external calcium is required for the enhancement of phosphatidylinositol turnover, (2) the accumulation of 45Ca by A-431 cells is stimulated by EGF, (3) blockage of calcium influx with LaCl3 inhibits stimulation of phosphatidylinositol turnover, and (4) calcium influx via ionophore A23187 is sufficient to stimulate phosphatidylinositol turnover. Since the binding, internalization, and degradation of 125I-labeled EGF in A-431 cells are unaffected by the omission of calcium from the medium, external calcium and phosphatidylinositol turnover are not necessary for the internalization and degradation of the EGF-receptor complex.

Protein-associated deoxyribonucleic acid strand breaks in L1210 cells treated with the deoxyribonucleic acid intercalating agents 4'-(9-acridinylamino) methanesulfon-m-anisidide and adriamycin.

Abstract: The DNA intercalating agents 4'-(9-acridinyl-amino) methanesulfon-m-anisidide (m-AMSA) and adriamycin were studied by using filter elution methods to measure DNA single-strand breaks (SSB's), DNA-protein cross-links (DPC's), and double-stranded breaks (DSB's) in mouse leukemia L1210 cells. Both compounds produced SSB's and DPC's at nearly 1:1 ratios. The SSB's and DPC's were shown to be localized with respect to each other; this was inferred from the finding that filter assays based on protein adsorption completely prevented the elution of the DNA single-strand segments between SSB's. In the case of m-AMSA, which produces relatively high frequencies of DNA lesions, the possibility that a protein bridges across the SSB was excluded by alkaline sedimentation studies. Both compounds also produced DSB's, but the SSB/DSB ratios differed; the SSB/DSB ratios increase in the following order: ellipticine greater than adriamycin greater than m-AMSA greater than X-ray [results of this paper combined with those of Ross, W. E., & Bradley, M. O. (1981) Biochim. Biophys. Acta (in press)]. The o-AMSA isomer is much less cytotoxic than m-AMSA and did not produce protein-associated strand breaks. The simplest model to explain the results is that a protein becomes covalently bound to either the 3' or the 5' termini of the intercalator-induced strand breaks. At moderately cytotoxic doses, m-AMSA yielded much larger frequencies of protein-associated SSB's than did adriamycin. m-AMSA-induced protein-associated SSB's saturated at approximately 60000 per cell over a concentration range in which m-AMSA uptake by the cells was proportional to the drug concentration. m-AMSA was found to enter and exit from cells very rapidly at 37 degrees C; protein-associated SSB's and DSB's also appeared and disappeared rapidly. At reduced temperature, however, the appearance and disappearance of protein-associated SSB's could be blocked while m-AMSA entry and exit still occurred. The saturation behavior and temperature dependence suggest that the formation and disappearance of protein-associated strand breaks is enzymatic. The simplest hypothesis is that the linked protein is a nuclease, such as a topoisomerase, which becomes bound to one terminus of the strand break it produces. It is proposed that topoisomerases producing SSB's and DSB's are stimulated to different degrees by different intercalators.

Taxol assembles tubulin in the absence of exogenous guanosine 5'-triphosphate or microtubule-associated proteins.

Abstract: Taxol increases the rate and extent of microtubule assembly in vitro and stabilizes microtubules in vitro and in cells [Schiff, P. B., Fant, J., & Horwitz, S. B. (1979) Nature (London) 277, 665-667; Schiff, P. B., & Horwitz, S. B. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 1561-1565]. We report herein that taxol has the ability to promote microtubule assembly in the absence of microtubule-associated proteins, rings, and added guanosine 5'-triphosphate (GTP or organic buffer. The drug enhances additional microtubule assembly when added to microtubules at apparent steady state. This additional assembly can be attributed to both elongation of existing microtubules and spontaneous nucleation of new microtubules. Taxol-treated microtubules have depressed dissociation reactions as determined by dilution experiments. The drug does not inhibit the binding of GTP or the hydrolysis of GTP or guanosine 5'-diphosphate (GDP) in our microtubule protein preparations. Taxol does not competitively inhibit the binding of colchicine to tubulin.

Mechanism of cholesterol and phosphatidylcholine exchange or transfer between unilamellar vesicles

Abstract: The mechanism of cholesterol and phosphatidylcholine exchange has been investigated by following the transfer of radiolabeled cholesterol and phosphatidylcholine from negatively charged, unilamellar cholesterol-egg yolk phosphatidylcholine donor vesicles to neutral acceptor vesicles of similar composition. Vesicles were incubated in the absence of protein and were stable to fusion over the course of the experiment. At intervals, donor and acceptor vesicles were separated by passage through a column of DEAE-Sepharose; less than 1% of the charged and 80-95% of the neutral vesicles were recovered in the eluate. Over 12 h at 37 degrees C, 90% of the donor vesicle [4-14C]cholesterol was transferred to the acceptor vesicles in a first-order process whose half-time was 2.3 +/- 0.3 h. This indicates that transfer of cholesterol molecules from the inner to outer monolayer of the vesicle bilayer is not rate limiting in exchange. In contrast to cholesterol exchange, the half-time for 1-palmitoyl-2-oleoyl[1-14C]phosphatidylcholine exchange was 48 +/- 5 h so that more than six molecules of cholesterol were transferred for each molecule of phosphatidylcholine. The interfacial flux of cholesterol from the donor bilayer is 5.3 x 10(-15) mol cm-2 s-1 (approximately 3 molecules/min for an average vesicle) and is similar to fluxes observed in other systems where phosphatidylcholine or cholesterol ester exchange is catalyzed by an exchange protein. When the acceptor vesicle concentration was increased 20-fold in cholesterol exchange experiments or 9-fold in phosphatidylcholine exchange experiments, the rate of label transfer was not affected. The activation energy of cholesterol exchange between 15 and 37 degrees C was 73 +/- 5 kJ mol-1. Transfer of cholesterol across a dialysis membrane is shown to be a slow process whose rate may be predicted by application of Fick's first law of diffusion. These results are only consistent with a mechanism of lipid exchange in which cholesterol and phosphatidylcholine diffuse through the aqueous phase; the experimental activation energy is associated with desorption of lipid from the donor bilayer into the aqueous phase.

Pyranine (8-hydroxy-1,3,6-pyrenetrisulfonate) as a probe of internal aqueous hydrogen ion concentration in phospholipid vesicles

Abstract: The fluorescence intensity (at 510 nm) of the hydrophilic pyrene analogue 8-hydroxy-1,3,6-pyrenetrisulfonate (pyranine) is strongly dependent upon the degree of ionization of the 8-hydroxyl group (pKa = 7.2) and hence upon the medium pH, over the range pH 6--10. Because of its polyanionic character, pyranine does not bind significantly to phospholipid vesicles having a net anionic surface charge. As a result, it is possible to form vesicles in the presence of pyranine which, after removal of external probe by gel filtration, contain pyranine entrapped within the internal aqueous compartment. Once entrapped, pyranine does not readily leak out of the vesicles. Because the fluorescence properties of entrapped pyranine resemble closely the properties of bulk pyranine solution with respect to pH sensitivity, pyranine can be used as a reliable reporter of aqueous pH changes within anionic vesicles. When HCl is rapidly added to a suspension of unilamellar soybean phospholipid (asolectin) vesicles preincubated at alkaline pH, a biphasic decrease in the pH of the vesicle inner aqueous compartment is observed. An initial, very rapid and electrically uncompensated H+ influx (t 1/2 less than 1 s) results in the generation of a transmembrane electric potential opposing further H+ influx. This leads to the development of a much slower (t 1/2 approximately equal to 5 min), valinomycin-sensitive, proton--counterion exchange which continues until the proton concentration gradient is eliminated. Similar results were obtained in asolectin vesicles prepared by detergent dilution, in sonicated egg phosphatidylcholine vesicles, and in multilamellar asolectin liposomes. The rather high permeability of soybean lipid membranes to H+ is surprising in view of the widespread use of these lipids for the reconstitution of membrane proteins which are thought to generate or utilize H+ ion gradients in energy transduction reactions.

Effects of cations on affinity of calmodulin for calcium: ordered binding of calcium ions allows the specific activation of calmodulin-stimulated enzymes. Theoretical approach to study of multiple ligand binding to a macromolecule

Abstract: The acid stability of calmodulin has been used to devise a rapid and efficient method of decalcification based on trichloroacetic acid precipitation. Study of the competitive binding of K+, Mg2+, and Ca2+ to the Ca2+-binding sites of calmodulin has allowed determination of the intrinsic binding constants of each of the three cations for the four Ca2+-binding sites. The data are compatible with an ordered binding of Ca2+. If the Ca2+ sites are labeled A, B, C, and D starting at the NH2 terminus, the order of binding is postulated to be B, A, C, and D. The ordered binding properties support the suggestion that calmodulin translates quantitative Ca2+ signals into qualitatively different cellular responses.

Calculations of enzymatic reactions: calculations of pKa, proton transfer reactions, and general acid catalysis reactions in enzymes.

Abstract: A method that allows one to correlate available X-ray data with activation free energies of enzymatic reactions is presented. This method is based on the empirical valence bond approach which uses experimental information to evaluate the energies of the valence bond resonance forms involved in a reaction and then calculates the environment-dependent stabilizations of the ionic resonance forms in the enzyme and in solution and correlates them with the rate acceleration by the enzyme. The method is reliable since it is based on calibration of potential surfaces by solution experiments and on transfer of the calibrated surface to the enzyme active site, using only simple calculations of electrostatic interactions. The close relation between the method and the intuitive valence bond description of bond-breaking bond-making reactions provides a new insight into enzymatic reactions, describing them as crossing between covalent and ionic valence bond resonance forms. Such a description correlates the stabilization of the ionic resonance forms by the enzyme active site with the enzyme catalytic activity. The paper considers the energetics of several enzymatic processes, including ionization, of acidic groups in enzyme active sites, stability of ion pairs in enzymes and in solutions, proton transfer reactions, and general acid catalysis reactions. The calculations support the idea that enzymes can be viewed as "supersolvents" that stabilize (solvate) ionic transition states more effectively than do aqueous solutions.

Electric field induced transient pores in phospholipid bilayer vesicles.

Abstract: A study of the voltage induction of transient pores in phospholipid bilayer vesicles is reported. Unilamellar vesicles (dipalmitoylphosphatidylcholine), with a size distribution of 100 +/- 30 nm, were prepared by the method of Enoch & Strittmatter [Enoch, H., & Strittmatter, P. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 145]. The vesicles loaded with [14C]sucrose and suspended in a mixture of 150 mM NaCl and 272 mM sucrose (both are the isotonic solvent for erythrocytes) were exposed to an intense electric field in the range of 20--40 kV/cm, with a field decay time of 5--15 micro second. A transient leakage of sucrose label was detected when the field strength exceeded 30 kV/cm. After the field was removed, no slow leakage of the tracer molecules occurred during a 65-h incubation period at the room temperature (23 +/- 2 degrees C). The leakage is attributed to the field-induced transmembrane potential, but not other effects such as the Joule heating or the shock wave associated with the voltage discharge. When the potential exceeded a threshold value of 200 mV, corresponding to an applied field strength of 30 kV/cm, there was a dielectric breakdown of the bilayer structure. Pores which allowed passage of sucrose were formed, transiently. Experiments show that these pores were fully reversible, and no global and permanent damages to the vesicle bilayer were detected. The implication of this membrane potential triggered conducting state of lipid bilayers to biological functions of cells is discussed.

Microsomal cytochrome P-450 from neonatal pig testis: two enzymatic activities (17 alpha-hydroxylase and c17,20-lyase) associated with one protein.

Abstract: Studies have been performed to test the hypothesis that cytochrome P-450 from testicular microsomes consists of a single protein with two enzymatic activities (17 alpha-hydroxylase and C17,20-lyase) Three lines of evidence to support the hypothesis were obtained (1) The enzyme appears to be homogeneous by immunochemical criteria with anti-P-450 IgG (line of identity on immunodiffusion and a single band on immunoelectrophoresis), by demonstration of a single NH2-terminal amino acid (methionine) and the finding of 16 single amino acids at the NH2 terminus (2) Optima for pH and temperature are the same for both enzymatic activities (pH 725 and 37 degrees C), and temperatures between 30 and 44 degrees C decreased both activities in such a way that the ratio of hydroxylase to lyase was the same at all temperatures tested (3) A variety of inhibitors affect both activities to the same extent: Ki values for two competitive inhibitors (SU 8000, 004 microM; SU 10603, 03 microM) are the same for hydroxylase and lyase; partition coefficients for inhibition by carbon monoxide are similar for hydroxylase and lyase (20 +/- 2 and 27 +/- 3); anti-P-450 (serum and IgG) causes inhibition of both activities to the same extent, and the same is true of a variety of less specific inhibitors It is concluded that a single heme protein (cytochrome P-450) from microsomes of neonatal pig testis catalyzes two reactions (hydroxylase and lyase) which are sequential steps in the synthesis of androgens by the testis leading to conversion of C21 precursors to C19 steroid hormones

Effect of macromolecular crowding upon the structure and function of an enzyme: glyceraldehyde-3-phosphate dehydrogenase

Abstract: The specific activity of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPD) has been measured as a function of GAPD concentration in the absence and presence of 18 g/dL ribonuclease A. The specific activity of GAPD at fixed concentration has been measured as a function of the concentration of added ribonuclease A, beta-lactoglobulin, bovine serum albumin, and poly(ethylene glycol) (Mr 20000) at additive concentrations of up to 30 g/dL. All of the data may be semiquantitatively accounted for by a simple model based upon the following qualitative assumptions: (1) Under the conditions of the reported experiments, GAPD exists primarily as an equilibrium mixture of monomers and tetramers of GAPD subunits. (2) The monomers have a much larger specific activity than do the tetramers. (3) The addition of high concentrations of unrelated globular proteins does not affect the activity of either monomer or tetramer but does promote the formation of tetramer due to space-filling properties of the added species, as proposed by Minton [Minton, A. P. (1981) Biopolymers (in press)].

Structure of ferric pseudobactin, a siderophore from a plant growth promoting Pseudomonas.

Abstract: Both plant growth promoting Pseudomonas B10 and its yellow-green, fluorescent iron transfer agent (siderophore) pseudobactin enhance the growth of the potato and control certain phytopathogenic microorganisms. The structure of the little compound has been determined by single-crystal X-ray diffraction methods using counter data. The structure consisted of a linear hexapeptide, L-Lys-D-threo-beta-OH-Asp-L-Ala-D-allo-Thr-L-Ala-D-N delta-OH-Orn, in which the N delta-OH nitrogen of the ornithine was cyclized with the C-terminal carboxyl group, and the N epsilon-amino group of the lysine was linked via an amide bond to a fluorescent quinoline derivative. The iron-chelating groups consisted of a hydroxamate group derived from N delta-hydroxyornithine, an alpha-hydroxy acid derived from beta-hydroxyaspartic acid, and an o-dihydroxy aromatic group derived from the quinoline moiety. The combination of metal-chelating ligands and the alternating L- and D-amino acids was unusual. The little compound crystallized as a single coordination isomer with the lambda absolute configuration. The present study is the first structural determination of a fluorescent siderophore. In the crystal structure, ferric pseudobactin formed a dimer, which constituted the asymmetric unit. The asymmetric unit also contained 26 water molecules. The molecules in the dimer were related by a pseudo-2-fold symmetry axis. Red-brown crystals of ferric pseudobactin (C42H57N12O16Fe . 13H2O), obtained from pyridine-acetic acid buffer solution equilibrated with water, conformed to space group I2 with a = 29.006 (23) A, b = 14.511 (13) A, c = 28.791 (21) A, and beta = 96.06 (5) degrees at -135 (2) degrees C. For eight molecules per unit cell, the calculated density was 1.38 g/cm3; the observed density was 1.40 g/cm3. The structure was refined by least-squares methods with anisotropic thermal parameters for all nonhydrogen atoms to a final R factor of 0.08 (8989 observed reflections).