Determination of molecular parameters by fitting sedimentation data to finite-element solutions of the Lamm equation.

A method for fitting experimental sedimentation velocity data to finite-element solutions of various models based on the Lamm equation is presented. The method provides initial parameter estimates and guides the user in choosing an appropriate model for the analysis by preprocessing the data with the G(s) method by van Holde and Weischet. For a mixture of multiple solutes in a sample, the method returns the concentrations, the sedimentation (s) and diffusion coefficients (D), and thus the molecular weights (MW) for all solutes, provided the partial specific volumes (v) are known. For nonideal samples displaying concentration-dependent solution behavior, concentration dependency parameters for s(sigma) and D(delta) can be determined. The finite-element solution of the Lamm equation used for this study provides a numerical solution to the differential equation, and does not require empirically adjusted correction terms or any assumptions such as infinitely long cells. Consequently, experimental data from samples that neither clear the meniscus nor exhibit clearly defined plateau absorbances, as well as data from approach-to-equilibrium experiments, can be analyzed with this method with enhanced accuracy when compared to other available methods. The nonlinear least-squares fitting process was accomplished by the use of an adapted version of the "Doesn't Use Derivatives" nonlinear least-squares fitting routine. The effectiveness of the approach is illustrated with experimental data obtained from protein and DNA samples. Where applicable, results are compared to methods utilizing analytical solutions of approximated Lamm equations.     

Impact of self-association on function of apolipoprotein A-I

Self-association is an inherent property of the lipid-free forms of several exchangeable apolipoproteins, including apolipoprotein A-I (apoA-I), the main protein component of high density lipoproteins (HDL) and an established antiatherogenic factor. Monomeric lipid-free apoA-I is believed to be the biologically active species, but abnormal conditions, such as specific natural mutations or oxidation, produce an altered state of self-association that may contribute to apoA-I dysfunction. Replacement of the tryptophans of apoA-I with phenylalanines (ΔW-apoA-I) leads to unusually large and stable self-associated species. We took advantage of this unique solution property of ΔW-apoA-I to analyze the role of self-association in determining the structure and lipid-binding properties of apoA-I as well as ATP-binding cassette A1 (ABCA1)-mediated cellular lipid release, a relevant pathway in atherosclerosis. Monomeric ΔW-apoA-I and wild-type apoA-I activated ABCA1-mediated cellular lipid release with similar efficiencies, whereas the efficiency of high order self-associated species was reduced to less than 50%. Analysis of specific self-associated subclasses revealed that different factors influence the rate of HDL formation in vitro and ABCA1-mediated lipid release efficiency. The α-helix-forming ability of apoA-I is the main determinant of in vitro lipid solubilization rates, whereas loss of cellular lipid release efficiency is mainly caused by reduced structural flexibility by formation of stable quaternary interactions. Thus, stabilization of self-associated species impairs apoA-I biological activity through an ABCA1-mediated mechanism. These results afford mechanistic insights into the ABCA1 reaction and suggest self-association as a functional feature of apoA-I. Physiologic mechanisms may alter the native self-association state and contribute to apoA-I dysfunction.     

Conformational studies of aqueous melittin: thermodynamic parameters of the monomer-tetramer self-association reaction

The self-association reaction in which four melittin molecules associate to form an aqueously soluble tetramer was studied by fluorescent spectroscopy. At 23 degrees C, pH 7.15, gamma/2 0.50, the dissociation constant, Kd, is 3.20 x 10(-16) M3. At 23 degrees C, gamma/2 0.60, melittin has an amino acyl group with a proton ionization constant at ca. 10(-6) M, which must be un-ionized for tetramer formation to occur. The change in Kd with temperature indicates the forward reaction (tetramer formation) proceeds primarily by entropic changes, with delta H degrees = -20.3 kJ/mol of monomer and delta S degrees = 211 J/(K . mol of monomer). The observed enthalpic and entropic values for the tetramerization reaction are consistent with the expected contributions of both nascent hydrogen bonds and hydrophobic stabilization to the reaction. The ionic strength dependence of the tetramerization reaction was found to be consistent with an Edsall-Wyman treatment of activity coefficients. Specifically, the calculated charge of melittin varied from 2.5 (pH 10.53, gamma/2 less than 0.08) to ca. 6 (pH 7.15, gamma/2 greater than 0.3) and showed a strong dependence on gamma/2.     

Molecular mass determination by sedimentation velocity experiments and direct fitting of the concentration profiles.

A new method for the direct molecular mass determination from sedimentation velocity experiments is presented. It is based on a nonlinear least squares fitting procedure of the concentration profiles and simultaneous estimation of the sedimentation and diffusion coefficients using approximate solutions of the Lamm equation. A computer program, LAMM, was written by using five different model functions derived by Fujita (1962, 1975) to describe the sedimentation of macromolecules during centrifugation. To compare the usefulness of these equations for the analysis of hydrodynamic results, the approach was tested on data sets of Claverie simulations as well as experimental curves of some proteins. A modification for one of the model functions is suggested, leading to more reliable sedimentation and diffusion coefficients estimated by the fitting procedure. The method seems useful for the rapid molecular mass determination of proteins larger than 10 kDa. One of the equations of the Archibald type is also suitable for compounds of low molecular mass, probably less than 10 kDa, because this model function requires neither the plateau region nor a meniscus free of solute.     

Specific inhibition of GPI-anchored protein function by homing and self-association of specific GPI anchors

The functional specificity conferred by glycophosphatidylinositol (GPI) anchors on certain membrane proteins may arise from their occupancy of specific membrane microdomains. We show that membrane proteins with noninteractive external domains attached to the same carcinoembryonic antigen (CEA) GPI anchor, but not to unrelated neural cell adhesion molecule GPI anchors, colocalize on the cell surface, confirming that the GPI anchor mediates association with specific membrane domains and providing a mechanism for specific signaling. This directed targeting was exploited by coexpressing an external domain-defective protein with a functional protein, both with the CEA GPI anchor. The result was a complete loss of signaling capabilities (through integrin-ECM interaction) and cellular effect (differentiation blockage) of the active protein, which involved an alteration of the size of the microdomains occupied by the active protein. This work clarifies how the GPI anchor can determine protein function, while offering a novel method for its modulation.     

Determination of the antenna heterogeneity of Photosystem II by direct simultaneous fitting of several fluorescence rise curves measured with DCMU at different light intensities

Several methods for determination of the antenna heterogeneity of Photosystem II from fluorescence rise curves measured with DCMU have been developed so far. Using these methods, two, three or four types of Photosystem II with respect to the antenna heterogeneity were determined. However, the accuracy of some of these methods is under debate. Here, we present a new method for the determination of the antenna heterogeneity of Photosystem II. The method is based on direct simultaneous fitting of several fluorescence rise curves measured with DCMU at different intensities of light excitation. As several curves measured under different light conditions are fitted simultaneously by the same model, reliability and accuracy in determination of model parameters increase. Our method was applied to two plant materials with different structure of the thylakoid membrane: wheat leaves and cells of green alga Chlamydomonas reinhardtii. This revised version was published online in June 2006 with corrections to the Cover Date.     

The role of self-association in Fin1 function on the mitotic spindle

Stabilization of spindle microtubules during anaphase is essential for proper chromosome segregation. Fin1 is a budding yeast protein that localizes to the poles and microtubules of the spindle during anaphase and contributes to spindle stability. The N-terminal half of Fin1 is phosphorylated at multiple sites by the cyclin-dependent kinase Clb5-Cdk1, and dephosphorylation in anaphase triggers its localization to the spindle. The C-terminal half of Fin1 contains coiled-coil motifs that are required for its self-association. Here we investigated the functional importance of the two regions of Fin1. Fin1 mutants lacking the C-terminal coiled-coil domains localized to spindle pole bodies but not along spindle microtubules. These mutants failed to self-associate and displayed reduced binding to microtubules in vitro but were functional in vivo and stabilized anaphase spindles when dephosphorylated. Deletion of the Fin1 C terminus suppressed the lethal phenotypes of the phospho-mutant Fin15A. Our findings suggest that the N-terminal region of Fin1 is sufficient for its regulated function as a spindle-stabilizing factor and that this function involves association with the spindle pole body. The ability of the C-terminal region to promote Fin1 self-association and microtubule binding may underlie the lethal effects of the deregulated Fin15A mutant.     

Curve fitting of germination data using the Richards function

Abstract. The fitting of the generalized Richards function to germination data by using two nested iterative and least squares regression procedures to estimate the four parameters (all of which can be associated with features of biological growth) is demonstrated. The program also involves a procedure of parallel curve analysis which makes comparisons between two curves by examining the whole process represented by the curve and not just a point or portion thereof. Excellent agreement between observed and expected values was obtained by analyzing data which defined patterns of germination exhibiting a range of rates and final percentages. The program also calculates a number of derived quantities including maximum daily rate of germination and time to 50% of final germination.     

Dietary effects of omega 3-fatty acids on intestinal transport function

Animals were fed for 2 weeks on one of four isocaloric and isocholesterolic semisynthetic diets: high 18:3 omega 3, low 18:3 omega 3, high 20:5 omega 3, or low 20:5 omega 3. The weight of the intestine and the percentage of the wall consisting of mucosa was greater in high 20:5 omega 3 than in high 18:3 omega 3, and greater in low 20:5 omega 3 than in low 18:3 omega 3, although the mucosal surface area was 26% lower in high 20:5 omega 3 than high 18:3 omega 3. The jejunal uptake of 40 mM glucose and ileal uptake of 40 mM galactose was greater in high 18:3 omega 3 than in high 20:5 omega 3, jejunal uptake of fatty acid 12:0 was higher, but 18:0 was lower in high 18:3 omega 3 than in high 20:5 omega 3. The jejunal or ileal uptake of cholesterol was not affected by 20:5 omega 3. However, 20:5 omega 3 had a variable effect on the uptake of medium- and long-chain fatty acids. Alterations in the uptake of fatty acids and glucose were not explained by any difference in the animals' food consumption, body weight gain, or intestinal weight, but the reduced jejunal uptake of 40 mM glucose in rats fed the high 20:5 omega 3 diet was associated with reduced mucosal surface area. Thus, (i) varying the source of omega 3-fatty acids (vegetable, 18:3 omega 3 versus fish oil, 20:5 omega 3) altered the mucosal mass of the intestine, and (ii) the source of the dietary omega 3-fatty acid (18:3 omega 3 versus 20:5 omega 3) influenced intestinal hexose uptake, with fish oil having an anti-absorptive effect on the jejunal uptake of D-glucose.     

Determination of chemical biodegradation by direct and indirect methods

Summary Degradation of 10 organic chemicals by pre-acclimated microorganisms in BOD dilution water was determined directly by UV spectrophotometry and indirectly by a modified BOD method. Residual chemical concentrations were periodically measured and pseudo-first-order biodegradation rate constants (k ₁) were calculated. Thek ₁ spectrophotometry values ranged from 0.006/h to 0.077/h andk ₁-BOD values from 0.002/h to 0.043/h for 1-methylnaphthalene and indole, respectively. The ratios ofk spectrophotometry to k₁-BOD were between 1.5 for salicylic acid and 3.0 for 1-methylnaphthalene with a mean of 2.7. A significant (=0.001) linear correlation (r ²=0.854,F=46.630) existed between the two sets of rate constants. Results from this study suggest that the modified BOD method may be used to estimate chemical biodegradation rates in synthetic media.     

Measurement of thermodynamic parameters for hydrophobic mismatch 1: self-association of a transmembrane helix

Membrane partitioning and self-association of transmembrane helices are crucial thermodynamic steps for membrane protein folding, although experimental difficulties have hampered quantitative estimations of related thermodynamic parameters, especially in lipid bilayer environments. This article reports for the first time, the complete set of thermodynamic parameters (DeltaG, DeltaH, DeltaS, and DeltaC(p)) for the formation of the antiparallel dimer of the inert hydrophobic model transmembrane helix X-(AALALAA)(3)-Y (X = 7-nitro-2-1, 3-benzoxadiazol-4-yl (NBD) and Y = NH(2) (I) or X = Ac and Y = NHCH(2)CH(2)-S-N-[4-[[4-(dimethylamino)phenyl]azo]phenyl]maleimide (DABMI) (II)) in dimonounsaturated phosphocholine lipid bilayers with different hydrophobic thicknesses (C14-C22) at 5-55 degrees C, as evaluated by fluorescence resonance energy transfer from I to II. Stronger dimerization was observed in thicker membranes and at lower temperatures (DeltaG = -9 to -26 kJ mol(-)(1)), driven by large negative DeltaH values (-18 to -80 kJ mol(-)(1)). Fourier transform infrared-polarized spectroscopy revealed that the peptide formed a stable transmembrane helix with an orientation angle of approximately 15 degrees in all bilayers without significant effects on lipid structures, suggesting that the depth to which the helix termini penetrate changes depending on the degree of hydrophobic mismatch. The enthalpy changes for helix-helix interactions can be well explained by the electrostatic interactions between helix macrodipoles in different dielectric environments. The new concept of dipole-dipole interaction as a basic driving force of helix dimerization will become a basis for understanding the structural and functional modifications in response to hydrophobic mismatch.     

Selective utilization of omega 6 and omega 3 polyunsaturated fatty acids by human skin fibroblasts

Incorporation of exogenous [14C] arachidonate by human skin fibroblasts was found to be significantly greater than that of either [14C]linoleate or alpha-[14C] linolenate. Arachidonate was preferentially esterified in the PI + PS and PE classes of phospholipids. Over 40% of the incorporated [14C] arachidonate was chain elongated in 24 hours. Cells were also grown in lipid-free medium to enhance PUFA desaturation and elongation and the utilization of various omega 6 and omega 3 metabolites examined. Whereas [14C] linoleate partitioned approximately 50:50 between PL and TAG, eicosatrienoate (20:3 omega 6) was selectively sequestered in TAG. Arachidonate and docosatetraenoate (22:4 omega 6) were preferentially incorporated into phospholipids; the PI + PS fraction was most highly enriched with arachidonate. Modification of alpha-[14C] linolenate was more extensive than that of [14C] linoleate. Docosapentaenoate (22:5 omega 3) was the major omega 3 [14C] PUFA of PI + PS and PE. Eicosapentaeonate was not selectively incorporated into phospholipids; within phospholipids the 20:5 omega 3 was primarily in PC. These results indicate that human skin fibroblasts exhibit acyl specificity in the esterification of polyunsaturated fatty acids, including preferential utilization of arachidonate rather than other prostaglandin precursors in the PI + PS fraction.     

Direct analysis of solute self-association by sedimentation equilibrium

An improved procedure is described for the characterization of solute self-association by sedimentation equilibrium. Whereas previous statistical-mechanical approaches to allowance for the effects of thermodynamic nonideality have entailed tedious iteration because of their specification of activity coefficients in terms of the equilibrium concentrations of all species, such reliance upon knowledge of the solution composition is avoided by the adaptation of an alternative statistical-mechanical formulation [T. L. Hill and Y. D. Chen (1973) Biopolymers, Vol. 12, pp. 1285–1312] in which thermodynamic nonideality is expressed in terms of total solute concentration. The development of an analysis in terms of a relationship with total solute concentration as the experimental variable allows this attribute of the Adams-Fujita approach to be retained without sacrifice of statistical-mechanical rigor. Its use is illustrated by application to Rayleigh interferometric records of sedimentation equilibrium distributions reflecting α-chymotrypsin dimerization and lysozyme self-association. © 1996 John Wiley & Sons, Inc.     

Assessment of GABARAP self-association by its diffusion properties

Gamma-aminobutyric acid type A receptor-associated protein (GABARAP) belongs to a family of small ubiquitin-like adaptor proteins implicated in intracellular vesicle trafficking and autophagy. We have used diffusion-ordered nuclear magnetic resonance spectroscopy to study the temperature and concentration dependence of the diffusion properties of GABARAP. Our data suggest the presence of distinct conformational states and provide support for self-association of GABARAP molecules. Assuming a monomer–dimer equilibrium, a temperature-dependent dissociation constant could be derived. Based on a temperature series of ¹H¹⁵N heteronuclear single quantum coherence nuclear magnetic resonance spectra, we propose residues potentially involved in GABARAP self-interaction. The possible biological significance of these observations is discussed with respect to alternative scenarios of oligomerization.