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PubMed2006

Analytical ultracentrifugation for the study of protein association and assembly.

Analytical ultracentrifugation remains pre-eminent among the methods used to study the interactions of macromolecules under physiological conditions. Recent developments in analytical procedures allow the high resolving power of sedimentation velocity methods to be coupled to sedimentation equilibrium approaches and applied to both static and dynamic associations. Improvements in global modeling based on numerical solutions of the Lamm equation have generated new sedimentation velocity applications with an emphasis on data interpretation using sedimentation coefficient or molar mass distributions. Procedures based on the use of multiple optical signals from absorption and interference optics for the analysis of the sedimentation velocity and equilibrium behavior of more complex interactions have now been developed. New applications of tracer sedimentation equilibrium experiments and the development of a fluorescence optical system for the analytical ultracentrifuge extend the accessible concentration range over several orders of magnitude and, coupled with the new analytical procedures, provide powerful new tools for studies of both weak and strong macromolecular interactions in solution.

Proteins↗

PubMed2004

Electrophoretic measurement of lipoprotein(a) cholesterol in plasma with and without ultracentrifugation: comparison with an immunoturbidimetric lipoprotein(a) method.

OBJECTIVES: Elevated plasma lipoprotein(a) [Lp(a)] is a significant risk factor for vascular disease. Standardization of Lp(a) mass measurement is complicated by the heterogeneity of apolipoprotein(a) [apo(a)]. We investigated whether Lp(a) cholesterol measurement, which is not influenced by apo(a) size, is a viable alternative to measuring Lp(a) mass. DESIGN AND METHODS: Plasma Lp(a) cholesterol was measured electrophoretically, with and without ultracentrifugation, and results were compared to each other and to immunoturbidimetrically measured Lp(a) mass in 470 subjects. RESULTS: Ultracentrifuged and whole plasma Lp(a) cholesterol levels demonstrated high correlation (R = 0.964). All samples with detectable (>/=2.0 mg/dl) Lp(a) cholesterol had Lp(a) mass >30 mg/dl (the clinically relevant cutpoint), while 59 samples with Lp(a) mass >30 mg/dl did not have detectable Lp(a) cholesterol. CONCLUSIONS: Lp(a) cholesterol can be measured in whole plasma without interference from VLDL lipoproteins. The relative clinical merits of measuring Lp(a) cholesterol vs. Lp(a) mass or both in combination deserves investigation.

Blotting, Western↗

PubMed2004

The dimeric and trimeric solution structures of the multidomain complement protein properdin by X-ray scattering, analytical ultracentrifugation and constrained modelling.

Properdin regulates the alternative pathway of the complement system of immune defence by stabilising the C3 convertase complex. It contains six thrombospondin repeat type I (TSR-1 to TSR-6) domains and an N-terminal domain. Properdin exists as either a dimer, trimer or tetramer. In order to determine the solution structure of multiple TSR domains, the molecular structures of dimeric and trimeric properdin were studied by X-ray scattering and analytical ultracentrifugation. Guinier analyses showed that the dimer and trimer have radii of gyration R(G) values of 7.5 nm and 10.3 nm, respectively, and cross-sectional radii of gyration R(XS) values of 1.3 nm and 1.5 nm, respectively. Distance distribution functions showed that the maximum lengths of the dimer and trimer were 25 nm and 30 nm, respectively. Analytical ultracentrifugation gave sedimentation coefficients of 5.1S and 5.2S for the dimer and trimer forms, respectively. Homology models for the TSR domains were constructed using the crystal structure of the TSP-2 and TSP-3 domains in human thrombospondin as templates. Properdin could be represented by seven TSR domains, not six as believed, since the crystal structure determined for TSP-2 and TSP-3 showed that the N-terminal domain (TSR-0) could be represented by a truncated TSR domain with the same six conserved Cys residues found in TSR-1 to TSR-6. Automated constrained molecular modelling revealed the solution conformations of multiple TSR domains in properdin at medium resolution. The comparison of 3125 systematically generated conformational models for the trimer with the X-ray data showed that good curve fits could be obtained by assuming that the linker between adjacent TSR domains possessed limited flexibility. Good trimer models correspond to partially collapsed triangular structures, and extended triangular shapes do not fit the data. The corresponding 3125 models for the dimer revealed a similar outcome in which a partially collapsed TSR structure gave good fits. The models account for the effect of mutations that cause properdin deficiencies, and suggest that the biologically active TSR-4, TSR-5 and TSR-6 domains are exposed for protein-protein interactions. The role of the other TSR domains in properdin may be to act as spacers to make TSR-4, TSR-5 and TSR-6 accessible for function.

Amino Acid Sequence↗

PubMed2005

Extended and flexible domain solution structure of the extracellular matrix protein anosmin-1 by X-ray scattering, analytical ultracentrifugation and constrained modelling.

Kallmann's syndrome corresponds to a loss of sense of smell and hypogonadotrophic hypogonadism. Defects in anosmin-1 result in the X-linked inherited form of Kallmann's syndrome. Anosmin-1 is an extracellular matrix protein comprised of an N-terminal, cysteine-rich (Cys-box) domain and a whey acidic protein-like (WAP) domain, followed by four fibronectin type III (FnIII) domains. The solution structures of recombinant proteins containing the first three domains (PIWF1) and all six domains (PIWF4) were determined by X-ray scattering and analytical ultracentrifugation. Guinier analyses showed that PIWF1 and PIWF4 have different radii of gyration (R(G)) values of 3.1 nm and 6.7 nm, respectively, but similar cross-sectional radii of gyration (R(XS)) values of 1.5 nm and 1.9 nm, respectively. Distance distribution functions showed that the maximum lengths of PIWF1 and PIWF4 were 11 nm and 23 nm, respectively. Analytical ultracentrifugation gave sedimentation coefficients of 2.52 S and 3.55 S for PIWF1 and PIWF4, respectively. The interpretation of the scattering data by constrained modelling requires homology models for all six domains in anosmin-1. While models were already available for the WAP and FnIII domains, searches suggested the Cys-box domain may resemble the cysteine-rich region of the insulin-like growth factor receptor. Automated constrained molecular modelling based on joining the anosmin-1 domains with structurally randomised linkers resulted in 10,000 models for anosmin-1. A trial-and-error search showed that about 0.1-1.4% of these models fitted the X-ray data. The best models showed that the three domains and six domains in PIWF1 and PIWF4, respectively, were extended. The inter-domain linkers in anosmin-1 could not all be extended at the same time, and there was evidence for inter-domain flexibility. Models with folded-back domain arrangements do not fit the data. These solution structures account for the known biological function of anosmin-1, in particular its ability to interact with its three macromolecular ligands.

Amino Acid Sequence↗

PubMed2005

Purification and characterization of retrovirus vector particles by rate zonal ultracentrifugation.

Sucrose equilibrium density ultracentrifugation remains the most widely used technique for retrovirus purification. However, purified virus preparations obtained by this routine method usually contain considerable amounts of contaminating cell membrane vesicles. In addition, sucrose solutions are highly viscous and hyperosmotic which jeopardizes the integrity and functionality of the retrovirus particle. In order to overcome these limitations, an alternative purification technique using rate zonal ultracentrifugation and iodixanol as gradient medium was developed. Recombinant retrovirus particles were produced by 293-GPG packaging cells grown in suspension in the presence of 10% FBS. Concentrated supernatants were purified by rate zonal sedimentation on a 10-30% continuous iodixanol gradient. Virus particles were recovered intact and active from the central fractions of the gradient. By using this strategy, high levels of purification were achieved, with no evident contamination with cell membrane vesicles as indicated by subtilisin treatment studies. The level of purity of the retrovirus preparation is over 95% as shown by SDS-PAGE analysis and size-exclusion chromatography. Purified particles appear homogenous in size and morphology according to negative stain electron microscopy. In addition, large amounts of defective retrovirus particles produced by 293-GPG packaging cells can be separated from functional retrovirus particles using this purification strategy.

Animals↗

PubMed2000

Ultracentrifuge and circular dichroism studies of folding equilibria in a retro GCN4-like leucine zipper.

Equilibrium ultracentrifuge and circular dichroism (CD) studies of a retropeptide of a GCN4-like leucine zipper in neutral saline buffer are reported as functions of temperature. Ultracentrifuge results indicate the presence of three oligomeric species: monomer, dimer, and tetramer, in quantifiable amounts, and the data provide values for the standard DeltaG, DeltaH, and DeltaS for interconversion. CD at 222 nm displays the strong concentration dependence characteristic of dissociative unfolding, but also shows a helicity far below that of the parent propeptide. Remarkably enough, the CD at 222 nm shows an extremum in the region between 0 and 20 degrees C. At higher T, the usual cooperative unfolding is observed. Comparable data are presented for a mutant retropeptide, in which a single asparagine residue is restored to the characteristic heptad position it occupies in the propeptide. The mutant shows marked differences from its unmutated relative in both thermodynamic properties and CD, although the oligomeric ensemble also comprises monomers, dimers, and tetramers. The mutant is closer in helicity to the parent propeptide but is less stable. These findings do not support either of the extant views on retropeptides. The behavior seen is consistent neither with the view that retropeptides should have the same structure as propeptides nor with the view that they should have the same structure but opposite chirality. The simultaneous availability of oligomeric population data and CD allows the latter to be dissected into individual contributions from monomers, dimers, and tetramers. This dissection yields explanations for the observed extrema in curves of CD (222 nm) versus T and reveals that the dimer population in both retropeptides undergoes "cold denaturation."

Amino Acid Sequence↗

PubMed1994

ESR studies of spin-labeled membranes aligned by isopotential spin-dry ultracentrifugation: lipid-protein interactions.

Electron spin resonance (ESR) studies have been performed on spin-labeled model membranes aligned using the isopotential spin-dry ultracentrifugation (ISDU) method of Clark and Rothschild. This method relies on sedimentation of the membrane fragments onto a gravitational isopotential surface with simultaneous evaporation of the solvent in a vacuum ultracentrifuge to promote alignment. The degree of alignment obtainable using ISDU, as monitored by ESR measurements of molecular ordering for both lipid (16-PC) and cholestane spin labels (CSL), in dipalmitoylphosphatidylcholine (DPPC) model membranes compares favorably with that obtainable by pressure-annealing. The much gentler conditions under which membranes may be aligned by ISDU greatly extends the range of macroscopically aligned membrane samples that may be investigated by ESR. We report the first ESR study of an integral membrane protein, bacteriorhodopsin (BR) in well-aligned multilayers. We have also examined ISDU-aligned DPPC multilayers incorporating a short peptide gramicidin A' (GA), with higher water content than previously studied. 0.24 mol% BR/DPPC membranes with CSL probe show two distinct components, primarily in the gel phase, which can be attributed to bulk and boundary regions of the bilayer. The boundary regions show sharply decreased molecular ordering and spectral effects comparable to those observed from 2 mol% GA/DPPC membranes. The boundary regions for both BR and GA also exhibit increased fluidity as monitored by the rotational diffusion rates. The high water content of the GA/DPPC membranes reduces the disordering effect as evidenced by the reduced populations of the disordered components. The ESR spectra obtained slightly below the main phase transition of DPPC from both the peptide- and protein-containing membranes reveals a new component with increased ordering of the lipids associated with the peptide or protein. This increase coincides with a broad endothermic peak in the DSC, suggesting a disaggregation of both the peptide and the protein before the main phase transition of the lipid. Detailed simulations of the multicomponent ESR spectra have been performed by the latest nonlinear least-squares methods, which have helped to clarify the spectral interpretations. It is found that the simulations of ESR spectra from CSL in the gel phase for all the lipid membranes studied could be significantly improved by utilizing a model with CSL molecules existing as both hydrogen-bonded to the bilayer interface and non-hydrogen-bonded within the bilayer.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1999

Direct sedimentation analysis of interference optical data in analytical ultracentrifugation.

Sedimentation data acquired with the interference optical scanning system of the Optima XL-I analytical ultracentrifuge can exhibit time-invariant noise components, as well as small radial-invariant baseline offsets, both superimposed onto the radial fringe shift data resulting from the macromolecular solute distribution. A well-established method for the interpretation of such ultracentrifugation data is based on the analysis of time-differences of the measured fringe profiles, such as employed in the g(s*) method. We demonstrate how the technique of separation of linear and nonlinear parameters can be used in the modeling of interference data by unraveling the time-invariant and radial-invariant noise components. This allows the direct application of the recently developed approximate analytical and numerical solutions of the Lamm equation to the analysis of interference optical fringe profiles. The presented method is statistically advantageous since it does not require the differentiation of the data and the model functions. The method is demonstrated on experimental data and compared with the results of a g(s*) analysis. It is also demonstrated that the calculation of time-invariant noise components can be useful in the analysis of absorbance optical data. They can be extracted from data acquired during the approach to equilibrium, and can be used to increase the reliability of the results obtained from a sedimentation equilibrium analysis.

Animals↗

PubMed2000

Interaction of different oligomeric states of hexameric DNA-helicase RepA with single-stranded DNA studied by analytical ultracentrifugation.

Analytical ultracentrifugation was used to determine the molecular mass, M, of hexameric DNA-helicase RepA at pH 5.8 and 7.6. At pH 7.6, a molecular mass of 179.5+/-2.6 kDa was found, consistent with the known hexameric state of RepA, (RepA)(6). At pH 5.8, (RepA)(6) associates to form a dimer with a molecular mass of 366.2+/-4.1 kDa. Analytical ultracentrifugation was also applied to characterize the interaction of single-stranded DNA (ssDNA) with the two different oligomeric states of (RepA)(6) at pH 5.8 and 7.6. The dissociation constants, K(d), for the equilibrium binding of (dA)(30) to the (RepA)(6) dimer at pH 5.8 and to (RepA)(6) at pH 7.6 were determined at 10 degrees C in the presence of 0.5 mM ATPgammaS, 10 mM MgCl(2) and 60 mM NaCl as K(d5.8)=0.94+/-0.13 microM at pH 5.8 and K(d7. 6)=25.4+/-6.4 microM at pH 7.6. The stoichiometries, n, for the two complexes (dA)(30)/(RepA)(6) dimer and (dA)(30)/(RepA)(6) at pH 5.8 and 7.6 were calculated from the corresponding binding curves. At pH 5.8 one (dA)(30) molecule was bound per (RepA)(6) dimer, while at pH 7.6 one (dA)(30) molecule was bound to one (RepA)(6). Binding curves were compatible with a single ssDNA binding site present on the (RepA)(6) dimer and on (RepA)(6), respectively, with no indication of cooperativity. (RepA)(6) tends to form larger aggregates under acidic conditions (pH<6.0) which are optimal for ssDNA binding. In contrast, at pH 5.8 in the presence of 60 mM NaCl, only the (RepA)(6) dimer was observed both in the absence and presence of (dA)(30).

Biopolymers↗

PubMed2000

Analytical ultracentrifugation in a Gibbsian perspective.

The analytical ultracentrifuge has come into new intensive use following complete instrumental redesign and the use of advanced computer technologies for the analysis and interpretation of experimental results. Major attention is now devoted to the evaluation of interactions between similar and dissimilar biological macromolecules in dilute and concentrated systems. Electrostatically charged biological solute systems additionally comprise low molecular weight charged and non-charged cosolvents. Solvent/cosolvent interactions, insufficiently considered in most current analytical ultracentrifugation analyses, may quantitatively affect solute/solute interactions. For comprehensive analysis the Svedberg derivation considering a buoyant molar mass (1 - rho0 partial specific volume)M2 and valid at vanishing solute concentration for strictly two component systems only, should be replaced, following classical thermodynamic analysis, by the ratio (delta rho/delta c2)(mu)/d pi/dc2 of the density increment at constant chemical potential of diffusible cosolvents, to the derivative of the osmotic pressure with solute concentration. Disregard of the solvent/cosolvent and solute/cosolvent interactions should be avoided.

Centrifugation, Density Gradient↗

PubMed2001

Biophysical studies by ultracentrifugation.

Advances in data analysis are broadening the applicability of ultracentrifugation to the characterization of macromolecular behavior in complex solution. The direct fitting of sedimentation velocity data to the Lamm equation is emerging as a very powerful means to characterize size distributions, improve the precision of data analysis and increase experimental throughput. With improvements in data acquisition and analysis, ultracentrifugation is poised to make significant contributions to our understanding of how macromolecules behave in vivo.

Biophysics↗

PubMed2001

Accurate separation of vesicles, micelles and cholesterol crystals in supersaturated model biles by ultracentrifugation, ultrafiltration and dialysis.

Gel filtration with bile salts at intermixed micellar/vesicular concentrations (IMC) in the eluant has been proposed to isolate vesicles and micelles from supersaturated model biles, but the presence of vesicular aggregates makes this method unreliable. We have now validated a new method for isolation of various phases. First, aggregated vesicles and - if present - cholesterol crystals are pelleted by short ultracentrifugation. Cholesterol contained in crystals and vesicular aggregates can be quantitated from the difference of cholesterol contents in the pellets before and after bile salt-induced solubilization of the vesicular aggregates. Micelles are then isolated by ultrafiltration of the supernatant through a highly selective 300 kDa filter and unilamellar vesicles by dialysis against buffer containing bile salts at IMC values. Lipids contained in unilamellar vesicles are also estimated by subtraction of lipid contents in filtered micelles from lipid contents in (unilamellar vesicle+micelle containing) supernatant ('subtraction method'). 'Ultrafiltration-dialysis' and 'subtraction' methods yielded identical lipid solubilization in unilamellar vesicles and identical vesicular cholesterol/phospholipid ratios. In contrast, gel filtration yielded much more lipids in micelles and less in unilamellar vesicles, with much higher vesicular cholesterol/phospholipid ratios. When vesicles obtained by dialysis were analyzed by gel filtration, vesicular cholesterol/phospholipid ratios increased strongly, despite correct IMC values for bile salts in the eluant. Subsequent extraction of column material showed significant amounts of lipids. In conclusion, gel filtration may underestimate vesicular lipids and overestimate vesicular cholesterol/phospholipid ratios, supposedly because of lipids remaining attached to the column. Combined ultracentrifugation-ultrafiltration-dialysis should be considered state-of-the-art methodology for quantification of cholesterol carriers in model biles.

Cholesterol↗

PubMed1993

5-hydroxytryptophan as a new intrinsic probe for investigating protein-DNA interactions by analytical ultracentrifugation. Study of the effect of DNA on self-assembly of the bacteriophage lambda cI repressor.

Pairwise cooperativity between proteins bound to DNA is believed to be important in governing the transcriptional regulation of numerous genes. However, the spectral overlap of normal proteins and DNA has blocked the study of these interactions by many physical methods. As shown recently by Ross et al. (in press), lambda cI repressor spectrally enhanced by 5-hydroxytryptophan (5-OHTrp), expressed in vivo using an Escherichia coli tryptophan auxotroph, exhibits dimer formation and DNA binding properties identical with those of the wild-type repressor. Moreover, the 5-OHTrp provides a spectral signal that allows monitoring of the protein concentration without interference from DNA. In this article, the ability to selectively detect 5-OHTrp-labeled repressor during analytical ultracentrifugation is used to study the higher order assembly of repressor dimers in the absence and in the presence of operator DNA. Contrary to the expectation that tetramer might be the limiting oligomer, lambda cI repressor undergoes a definite association to octamer. The relatively narrow concentration range over which transition from predominantly dimer to predominantly octamer occurs makes it unlikely that significant levels of tetramer are formed in the absence of DNA. Moreover, mass measurements reveal that an OR1 oligonucleotide binds to octameric repressor and does not dissociate it to tetramers. The use of the 5-OHTrp spectral enhancement opens a promising new avenue for the exploration of protein-protein and protein-nucleic acid interactions by analytical ultracentrifugation.

5-Hydroxytryptophan↗

PubMed1991

Self-association of human and porcine relaxin as assessed by analytical ultracentrifugation and circular dichroism.

The self-association properties of recombinant DNA derived human relaxin, and porcine relaxin isolated from porcine ovaries, have been studied by sedimentation equilibrium analytical ultracentrifugation and circular dichroism (CD). The human relaxin ultracentrifuge data were adequately defined by a monomer-dimer self-association model with an association constant of approximately 6 x 10(5) M-1, whereas porcine relaxin was essentially monomeric in solution. An approximate 5-fold increase in weight fraction of human relaxin monomer elicited by dilution of the protein resulted in no change in the far-UV CD spectrum at 220 nm. In contrast, after the same increase in weight fraction of monomer, the near-UV circular dichroism spectra for human relaxin exhibited a significant decrease in the amplitude for the CD bands near 277 and 284 nm. These CD bands, which may be assigned to the lone tyrosine in human relaxin, are superimposed on a broad envelope that is probably due to the three disulfide chromophores. Although both the human and porcine proteins contain two tryptophan residues, the near-UV CD spectra exhibit only a broad shoulder near 295 nm rather than the strong CD bands often found for tryptophan. Moreover, there is little change in this broad band after dilution of human relaxin to concentrations that resulted in a 4-fold increase in monomer weight fraction. These data suggest that dissociation of the human relaxin dimer to monomer is not accompanied by large overall changes in secondary structure or alteration in the average tryptophan environment, whereas there is a significant change in the tyrosine environment.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1985

Rabbit skeletal myosin heads in solution, as observed by ultracentrifugation and freeze-fracture electron microscopy: dimerization and maximum chord.

The use of analytical ultracentrifugation and freeze-fracture electron microscopy in solution allowed us to observe the monomeric and dimeric forms of Mg.71. This subfragment of the myosin molecule contains the LC2 light chain and is comparable to a "native" myosin head. Sedimentation-diffusion equilibrium ultracentrifugation shows that it is necessary to use slightly different conditions in order to obtain a pure Mg.S1 dimer, as compared to the case of chymotryptic S1 (LC2-free S1). For example, in a buffer leading to a complete dimerization of chymotryptic S1, Mg.S1 is only in the form of a monomer-dimer mixture, with comparable proportions of monomer and dimer. The freeze-fracture technique, applied to solutions containing Mg.S1 or chymotryptic S1, revealed that the monomeric species both have the same maximum chord (about 120 A) and that both dimeric species also have the same maximum chord (about 250 A). The maximum chord of the monomer is comparable to the surface-to-surface spacing between the myosin and actin filaments, in a fiber at the slack length. In sharp contrast this chord is higher than this spacing in a stretched fiber. The consequences of this fact are discussed, with particular reference to the sarcomere length-tension relationship.

Animals↗

PubMed2005

Size determination of cyanobacterial and higher plant photosystem II by gel permeation chromatography, light scattering, and ultracentrifugation.

The oxygen-evolving photosystem II core complexes (PSIIcc) from the thermophilic cyanobacterium Thermosynechococcus elongatus (PSIIccTe) and the higher plant Spinacia oleracea (PSIIccSo) have been isolated from the thylakoid membrane by solubilization with n-dodecyl-beta-d-maltoside, purified and characterized by gel permeation chromatography (GPC), dynamic light scattering (DLS), and analytical ultracentrifugation (AUC). DLS suggests that PSIIcc from both organisms exists as a monomer in dilute solution and aggregates with increasing protein concentration. In contrast to DLS, GPC and AUC showed that PSIIcc of both organisms occur as monomers and dimers, and it became clear from our studies that calibration of GPC columns with soluble proteins leads to wrong estimates of the molecular masses of membrane proteins. At a PSIIcc protein concentration of 0.2 mg/mL, molar masses, M, of 756 +/- 18 kDa and 710 +/- 15 kDa for dimeric PSIIccTe and PSIIccSo, respectively, were determined by analytical ultracentrifugation. At very low protein concentrations, at or below 0.05 mg/mL, the dimeric form of PSIIccTe partially dissociates (20-30%) to form monomers. On the basis of these studies 3-dimensional crystals of PSIIccTe were obtained that contain dimers in the asymmetric unit [Zouni, A. et al. (2001) Nature 409, 739-743]. Using synchrotron radiation the crystals diffract to a resolution of 3.8 A, which has been improved recently to 3.2 A [Biesiadka, J., et al. (2004) Phys. Chem. Chem. Phys. 6, 4733-4736].

Bacterial Proteins↗

PubMed2002

Secondary structural studies of bovine caseins: structure and temperature dependence of beta-casein phosphopeptide (1-25) as analyzed by circular dichroism, FTIR spectroscopy, and analytical ultracentrifugation.

The defining structural feature of all of the caseins is their common phosphorylation sequence. In milk, these phosphoserine residues combine with inorganic calcium and phosphate to form colloidal complexes. In addition, nutritional benefits have been ascribed to the phosphopeptides from casein. To obtain a molecular basis for the functional, chemical, and biochemical properties of these casein peptides, the secondary structure of the phosphopeptide of bovine beta-casein (1-25) was reexamined using Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopies. Both methods predict secondary structures for the peptide which include polyproline II elements as well as beta-extended sheet and turn-like elements. These structural elements were highly stable from 5 degrees to 70 degrees C. Reexamination of previously published 1H NMR data using chemical shift indices suggests structures in accord with the CD and FTIR data. Dephosphorylation showed little or no secondary structural changes, as monitored by CD and FTIR, but the modified peptide demonstrated pronounced self-association. The polymers formed were not highly temperature sensitive, but were pressure sensitive as judged by analytical ultracentrifugation at selected rotor speeds. Molecular dynamics (MD) simulations demonstrated relatively large volume changes for the dephosphorylated peptide, in accord with the pressure dependent aggregation observed in the analytical ultracentrifuge data. In contrast the native peptide in MD remained relatively rigid. The physical properties of the peptide suggest how phosphorylation can alter its biochemical and physiological properties.

Amino Acid Sequence↗

PubMed1971

Phosphatidylcholine-cholesterol acyltransferase in the ultracentrifugal residual protein fraction of rat plasma.

1. The phosphatidylcholine-cholesterol acyltransferase of rat plasma was dissociated from the plasma lipoproteins by ultracentrifugation and shown to be present in the plasma residual-protein fraction of density >1.210. 2. The general properties of the acyltransferase were substantially unchanged in the residual fraction as judged from the effects of differences in the substrates and of overnight starvation on the formation of different cholesterol esters. 3. The enzyme from rats starved overnight, by comparison with the enzyme from fed rats, preferentially formed cholesteryl arachidonate at the expense of cholesteryl linoleate. 4. The results suggest that ultracentrifugal separation of the plasma residual fraction may be used as an initial step for the purification of the acyltransferase.

Acyltransferases↗