Strategies for the deconvolution of diffusion in the determination of size-distributions from sedimentation velocity experiments were examined and developed. On the basis of four different model systems, we studied the differential apparent sedimentation coefficient distributions by the time-derivative method, g(s*), and by least-squares direct boundary modeling, ls-g*(s), the integral sedimentation coefficient distribution by the van Holde-Weischet method, G(s), and the previously introduced differential distribution of Lamm equation solutions, c(s). It is shown that the least-squares approach ls-g*(s) can be extrapolated to infinite time by considering area divisions analogous to boundary divisions in the van Holde-Weischet method, thus allowing the transformation of interference optical data into an integral sedimentation coefficient distribution G(s). However, despite the model-free approach of G(s), for the systems considered, the direct boundary modeling with a distribution of Lamm equation solutions c(s) exhibited the highest resolution and sensitivity. The c(s) approach requires an estimate for the size-dependent diffusion coefficients D(s), which is usually incorporated in the form of a weight-average frictional ratio of all species, or in the form of prior knowledge of the molar mass of the main species. We studied the influence of the weight-average frictional ratio on the quality of the fit, and found that it is well-determined by the data. As a direct boundary model, the calculated c(s) distribution can be combined with a nonlinear regression to optimize distribution parameters, such as the exact meniscus position, and the weight-average frictional ratio. Although c(s) is computationally the most complex, it has the potential for the highest resolution and sensitivity of the methods described.

The cytosolic C-terminal domain of the membrane copper transporter Ctr1 from the yeast Saccharomyces cerevisiae, Ctr1c, was expressed in E. coli as an oxygen-sensitive soluble protein with no significant secondary structure. Visible-UV spectroscopy demonstrated that Ctr1c bound four Cu(I) ions, structurally identified as a Cu(I)(4)(micro-S-Cys)(6) cluster by Xray absorption spectroscopy. This was the only metalated form detected by electrospray ionization mass spectrometry. An average dissociation constant K(D) = (K(1)K(2)K(3)K(4))(1/4) = 10(-)(19) for binding of Cu(I) to Ctr1c was estimated via competition with the ligand bathocuproine disulfonate bcs (beta(2) = 10(19.8)). Equivalent experiments for the yeast chaperone Atx1 and an N-terminal domain of the yeast Golgi pump Ccc2, which both bind a single Cu(I) ion, provided similar K(D) values. The estimates of K(D) were supported by independent estimates of the equilibrium constants K(ex) for exchange of Cu(I) between pairs of these three proteins. It is apparent that, in vitro, the three proteins buffer "free" Cu(I) concentrations in a narrow range around 10(-)(19) M. The results provide quantitative support for the proposals that, in yeast, (a) "free" copper concentrations are very low in the cytosol and (b) the Cu(I) trafficking gradient is shallow along the putative Ctrlc --> Atx1 --> Ccc2n metabolic pathway. In addition, both Ctr1c and its copper-responsive transcription factor Mac1 contain similar clusters which may be important in signaling copper status in yeast.

The specific self-association of proteins to form amyloid fibrils is a characteristic of a number of pathologies, including Alzheimer, Parkinson, and Creutzfeldt-Jakob diseases (1). This process involves slow nucleation coupled to self-association steps, which constitute an alternative folding pathway to those leading to the native state (2, 3). Amyloid formation is promoted by destabilization of the native state through events such as mutation or truncation. For example, certain mutants of lysozyme that exhibit molten globule characteristics also form amyloid (4) whereas apomyoglobin forms amyloid under partially denaturing conditions (5).Human apolipoprotein C-II (apoC-II) 1 (M r ϭ 8915, 79 residues) is normally a component of very low density lipoprotein, where it plays an important physiological role as an activator of lipoprotein lipase (6, 7). When associated with polar lipids (e.g. phospholipids or SDS) apoC-II adopts a primarily ␣-helical conformation (8 -10). Our previous work (11) demonstrates that, in the absence of lipid, human apoC-II self-associates to form twisted ribbon-like fibrils with all of the hallmarks of amyloid, including binding to Congo Red with red-green birefringence under cross-polarized light, binding to thioflavin T, and increased ␤-structure. In addition, x-ray diffraction patterns of aligned apoC-II fibrils indicate a cross-␤-sheet structure.2 In vitro amyloid formation by apoC-II can be compared with the in vivo deposition of amyloid involving other apolipoproteins such as apoA-I (12, 13), apoA-II (14, 15), apoA-IV (16), apoE (17), and apolipoprotein-like proteins, ␣-synuclein (18) and serum amyloid A (19). A significant clue to the prevalence of apolipoproteins in amyloid formation is provided by the observation that many apolipoproteins have limited conformational stability or secondary structure in the absence of lipid (20). Destabilized conformations in many proteins promote amyloid formation (3-5, 21). Apolipoprotein derivatives that form amyloid are frequently mutant isoforms or truncated products; for example, amyloid deposition involving apoA-I (12), apoA-II (14), and the C-terminal domain of apoE (17). We propose that these modifications destabilize lipid binding leading to amyloid formation in vivo (20). The well-characterized ability of apoC-II to form amyloid fibrils provides a convenient model to examine in vivo parameters (9, 22) that could control the growth of amyloid fibrils in vivo.Most biological fluids contain a high total concentration of macromolecules, including proteins, nucleic acids, and carbohydrates, that collectively occupy a high fraction of the fluid volume (23). Volume exclusion or "macromolecular crowding" in such fluids may result in significant alterations of the rates and equilibria of macromolecular associations (23-26). It has been suggested on conceptual grounds that volume exclusion in physiological media could modulate the rate and extent of amyloid formation in vivo (27,28). In the present study we investigate the potential effects of ma...

Shear flow is indirectly implicated in amyloid formation in vitro. Despite the association between amyloid fibrils and disease, and the prevalence of flow in physiological systems, the effect of this parameter is uncharacterized. We designed a novel Couette cell to quantitatively investigate shear exposure during fibrillogenesis. Amyloid formation by beta-lactoglobulin was monitored in situ with real-time fluorescence measurements across a range of shear rates. We demonstrate shear-induced aggregation of spheroidal seed-like species. These seeds enhance fibril formation in native beta-lactoglobulin, thereby demonstrating that shear flow generates an amyloidogenic precursor. Furthermore, preformed fibrils are degraded by exposure to high shear rates. Our results have implications for the mechanism of amyloid formation in physiological flow conditions.

Human apolipoprotein C-II (apoC-II) self-associates in solution to form aggregates with the characteristics of amyloid including red-green birefringence in the presence of Congo Red under cross-polarized light, increased fluorescence in the presence of thioflavin T, and a fibrous structure when examined by electron microscopy. ApoC-II was expressed and purified from Escherichia coli and rapidly exchanged from 5 M guanidine hydrochloride into 100 mM sodium phosphate, pH 7.4, to a final concentration of 0.3 mg/mL. This apoC-II was initially soluble, eluting as low molecular weight species in gel filtration experiments using Sephadex G-50. Circular dichroism (CD) spectroscopy indicated predominantly unordered structure. Upon incubation for 24 h, apoC-II self-associated into high molecular weight aggregates as indicated by elution in the void volume of a Sephadex G-50 column, by rapid sedimentation in an analytical ultracentrifuge, and by increased light scattering. CD spectroscopy indicated an increase in beta-sheet content, while fluorescence emission spectroscopy of the single tryptophan revealed a blue shift and an increase in maximum intensity, suggesting repositioning of the tryptophan into a less polar environment. Electron microscopy of apoC-II aggregates revealed a novel looped-ribbon morphology (width 12 nm) and several isolated closed loops. Like all of the conserved plasma apolipoproteins, apoC-II contains amphipathic helical regions that account for the increase in alpha-helix content on lipid binding. The increase in beta-structure accompanying apoC-II fibril formation points to an alternative folding pathway and an in vitro system to explore the general tendency of apolipoproteins to form amyloid in vivo.