Modern Applications of Analytical Ultracentrifugation

Laue, Thomas M.; Stafford, Walter F.

doi:10.1146/annurev.biophys.28.1.75

Cited by 259 publications

(202 citation statements)

References 124 publications

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“…(6) Values in parentheses correspond to the same simulation with fitting range slightly extended to larger maximal radii from 7.121 cm to 7.140 cm. (7) Simulated conditions resemble the experiments in…”

Section: Discussionmentioning

confidence: 89%

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Sedimentation equilibrium analysis of protein interactions with global implicit mass conservation constraints and systematic noise decomposition

Vistica¹,

Dam²,

Balbo³

et al. 2004

Analytical Biochemistry

333

396

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Sedimentation equilibrium is a powerful tool for the characterization of protein self-association and heterogeneous protein interactions. Frequently, it is applied in a configuration with relatively long solution columns and with equilibrium profiles being acquired sequentially at several rotor speeds.The present study proposes computational tools, implemented in the software SEDPHAT, for the global analysis of equilibrium data at multiple rotor speeds, multiple concentrations, and multiple optical detection methods. The detailed global modeling of such equilibrium data can be a non-trivial computational problem. It was shown previously that mass conservation constraints can significantly improve and extend the analysis of heterogeneous protein interactions. Here, a method for using conservation of mass constraints for the macromolecular redistribution is proposed in which the effective loading concentrations is being calculated from the sedimentation equilibrium profiles. The approach is similar to that described by Roark (Biophys. Chem. 5 (1976) [185][186][187][188][189][190][191][192][193][194][195][196], but its utility is extended by determining the bottom position of the solution columns from the macromolecular redistribution. For analyzing heterogeneous associations at multiple protein concentrations, additional constraints are introduced that relate the effective loading concentrations of the different components or their molar ratio in the global analysis. Equilibrium profiles at multiple rotor speeds also permit the algebraic determination of radial-dependent baseline profiles, which can govern interference optical ultracentrifugation data, but usually also occur, to a smaller extent, in absorbance optical data. Finally, the global analysis of equilibrium profiles at multiple rotor speeds with implicit mass conservation and computation of the bottom of the solution column provides an unbiased scale for determining molar mass distributions of non-interacting species. The properties of these tools are studied with theoretical and experimental data sets.

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Section: Discussionmentioning

confidence: 89%

“…Usually no protein modification is necessary. Recent reviews, and introductory texts on principle and practice can be found in (3)(4)(5)(6)(7)(8)(9)(10)(11)(12).…”

Section: Introductionmentioning

confidence: 99%

Sedimentation equilibrium analysis of protein interactions with global implicit mass conservation constraints and systematic noise decomposition

Vistica¹,

Dam²,

Balbo³

et al. 2004

Analytical Biochemistry

333

396

View full text Add to dashboard Cite

show abstract

“…Baseline correction, noise reduction and ellipticity calculations for the scans were done using Jasco J-700 for Windows Analytical Ultracentrifugation. Sedimentation equilibrium experiments were conducted at 30 °C in a Beckman XL-I Analytical Ultracentrifuge using interference optics, as described by Laue and Stafford (12). Aliquots (110 ul) of sample solution were loaded into 6-sector CFE sample cells, allowing 3 concentrations of sample to be run simultaneously.…”

Section: Methodsmentioning

confidence: 99%

Structure−Function Studies of Human Apolipoprotein A-V: A Regulator of Plasma Lipid Homeostasis

et al. 2003

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To investigate structure and function relations of a new member of the exchangeable apolipoprotein family that modulates plasma lipid levels, recombinant human apolipoprotein (apo) A-V was produced in Escherichia coli and isolated by a combination of nickel chelation affinity chromatography and reversed-phase HPLC. Antibodies directed against apoA-V were generated and employed in immunoblotting experiments. Anti-apoA-V IgG gave a strong response against recombinant apoA-V from E. coli and human apoA-V expressed in transgenic mice, but did not recognize human apoA-I or apoA-IV. In neutral-pH buffers, at concentrations of >0.1 mg/mL, isolated lipid-free apoA-V is poorly soluble. By contrast, apoA-V is soluble in 50 mM sodium citrate (pH 3.0). Far-UV circular dichroism analysis and spectral deconvolution reveal that apoA-V possesses 32% α-helix, 33% β-sheet, 16% β-turn, and 18% random coil secondary structure conformers. Temperature-induced denaturation studies gave rise to a transition midpoint of 47.1 °C. Upon being cooled to ambient temperature from 85 °C, apoA-V failed to recover all of the negative ellipticity present in unheated apoA-V. ApoA-V interacts with bilayer vesicles of dimyristoylphosphatidylcholine to form discoidal complexes with diameters in the range of 15−20 nm. However, apoA-V was a poor activator of lecithin:cholesterol acyltransferase where the activity was 8.5 ± 1.8% of that of apoA-I. Furthermore, apoA-V failed to support enhanced efflux of cholesterol from cAMP-treated J774 macrophages, although low levels of efflux were obtained from unstimulated cells. Taken together, the results demonstrate recombinant apoA-V possesses unique structural and functional characteristics, in keeping with its proposed role in the modulation of plasma lipid levels.

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“…At sedimentation equilibrium, the absorbance at a specified wavelength and position in the solution column is given by eq 1 (26,27).…”

Section: Host-guest Experimentsmentioning

confidence: 99%

Host−Guest Study of Left-Handed Polyproline II Helix Formation

et al. 2001

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The importance of the left-handed polyproline II (PPII) helical conformation has recently become apparent. This conformation generally is involved in two important functions: protein-protein interactions and structural integrity. PPII helices play vital roles in a variety of processes including signal transduction, transcription, and cell motility. Proline-rich regions of sequence are often assumed to adopt this structure. Remarkably, little is known about the physical determinants of this secondary structure type. In this study, we have explored the formation of PPII helices by a short poly(proline) peptide. In addition, the results from experiments used to determine the propensities for apolar residues, plus glycine, asparagine, and glutamine, to adopt this structure in a poly(proline)-based host peptide are reported here. Proline possesses the highest intrinsic propensity, with glutamine, alanine, and glycine having surprisingly high propensities.-Branched residues possess the lowest propensities of the residues examined. It is postulated that propensities possessed by apolar residues are due in part to peptide-solvent interactions, and that the remarkably high propensity possessed by glutamine may be due to a side chain to backbone hydrogen bond. These data are the first step toward a molecular understanding of the formation of this important, and yet little studied, secondary structure.In recent years, the left-handed polyproline II (PPII) 1 helical conformation has been elevated from the status of a relatively rare and seemingly uninteresting secondary structure to one that is surprisingly common and of the utmost importance. This structure plays a central role in numerous vital processes including signal transduction, transcription, cell motility, and the immune response. Proline-rich ligands of the cytoskeletal protein profilin (1), as well as those of the SH3, WW, and EVH1 protein interaction domains, are bound in this conformation (2). The peptide ligands of class II MHC molecules are also bound in the PPII conformation (3). PPII helices are major features of collagens (4) and plant cell wall proteins (5). The PPII helix is believed to be the dominant conformation for many proline-rich regions of sequence (PRRs) (6). Sequences not rich in proline also adopt this structure. For example, poly(lysine), poly(glutamate), and poly(aspartate) peptides form PPII helices (7). Around 2% of all residues in known protein structures are found in PPII helices at least four residues long (8, 9). As many as 10% of all residues are found in the PPII conformation, although not necessarily as part of PPII helices (10). PPII helices have also been hypothesized to be a major component of protein denatured states (11-14), giving them a role in a most fundamental process. Recently, Blanch et al. (15) have suggested that the PPII helix might be the precursor conformation in amyloid formation. Given the preceding, it is truly remarkable how little is known about the physical determinants of the PPII helical conformat...

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Modern Applications of Analytical Ultracentrifugation

Cited by 259 publications

References 124 publications

Sedimentation equilibrium analysis of protein interactions with global implicit mass conservation constraints and systematic noise decomposition

Sedimentation equilibrium analysis of protein interactions with global implicit mass conservation constraints and systematic noise decomposition

Structure−Function Studies of Human Apolipoprotein A-V: A Regulator of Plasma Lipid Homeostasis

Host−Guest Study of Left-Handed Polyproline II Helix Formation

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