Asaf Grupi

Capillary Viscometer for Fully Automated Measurement of the Concentration and Shear Dependence of the Viscosity of Macromolecular Solutions

The construction and operation of a novel viscometer/rheometer are described. The instrument is designed to measure the viscosity of a macromolecular solution while automatically varying both solute concentration and shear rate. Viscosity is calculated directly from Poiseuilles law, given the measured difference in pressure between two ends of a capillary tube through which the solution is flowing at a known rate. The instrument requires as little as 0.75 mL of a solution to provide a full profile of viscosity as a function of concentration and shear rate, and it can measure viscosities as high as 500 cP and as low as 1 cP, at shear rates between 10 and 2 × 10(3) s(-1). The results of control experiments are presented to document the accuracy and precision of measurement at both low and high concentration of synthetic polymers and proteins.

The obese phenotype-inducing N82K mutation in human leptin disrupts receptor-binding and biological activity.

A novel homozygous mutation of the leptin gene was recently reported in an Egyptian child and his sister with severe early onset obesity. This mutation results from the substitution of asparagine (AAC) by lysine (AAA) at codon 103 of a non-mature (signal peptide-containing) leptin and corresponds to the N82K mutation in the mature protein. The patient had very low serum leptin levels, raising the question of whether the obese phenotype resulted from low leptin levels or from its lower intrinsic activity. To answer this question, we characterized the functional consequences of the N82K mutation. Wild-type (WT) human leptin was mutated accordingly, expressed in Escherichia coli at high yield, purified to homogeneity as a monomer and compared to WT human leptin prepared by the same methodology. Circular dichroism analysis of the mutated leptin indicated proper refolding and a secondary structure identical to that of the WT human leptin. In contrast to WT human leptin, the N82K mutant did not form a detectable complex with human leptin-binding domain (hLBD) and its binding capacity to hLBD assessed in a nonradioactive receptor-binding assay was at least 500-fold lower than that of WT human leptin. The biological activity of the N82K mutant, tested in two cell bioassays, was reduced by more than three orders of magnitude relative to WT human leptin. Therefore, though the present report does not explain the reason for the low circulating leptin levels it definitely documents that the reported obese phenotype originates not only from low serum leptin levels but also from the N82K mutants almost total lack of intrinsic leptin activity.

Concentration-Dependent Viscosity of Binary and Ternary Mixtures of Nonassociating Proteins: Measurement and Analysis

Using a recently developed automated viscometer (Grupi, A.; Minton, A. P. Anal. Chem. 2012, 84, 10732-10736), the dependence of solution viscosity upon the concentrations of bovine serum albumin, hen egg ovomucoid, and human fibrinogen have been measured individually and in binary and ternary mixtures over a wide range of compositions and at total concentrations of up to 300 g/L. The concentration dependence of viscosity of all solutions is quantitatively described over the entire range of concentrations and compositions by a semiempirical equation requiring specification of only two composition-independent global parameters per protein.

Transient secondary and tertiary structure formation kinetics in the intrinsically disordered state of α-Synuclein from atomistic simulations.

In the absence of a stable fold, transient secondary structure kinetics define the native state of the prototypical and pharmacologically relevant intrinsically disordered protein (IDP) α-Synuclein (aS). Here, we investigate kinetics preventing ordering and possibly pathogenic β-sheet aggregation. Interestingly, transient β-sheets form frequently at sub μs time scales precisely at the positions observed in aS amyloid fibrils. The formation kinetics competes with rapid secondary structure dissociation rates, thus explaining the low secondary structure content. The fast secondary structure dissociation times are very similar to the dynamics of tertiary structure rearrangements. These findings suggest that the fast dissociation kinetics slows down conformational selection processes for aS aggregation, which may be a general mechanism controlling the aggregation kinetics of IDPs.

Intramolecular Diffusion in α-Synuclein: It Depends on How You Measure It

Intramolecular protein diffusion, the motion of one part of the polypeptide chain relative to another part, is a fundamental aspect of protein folding and may modulate amyloidogenesis of disease-associated intrinsically disordered proteins. Much work has determined such diffusion coefficients using a variety of probes, but there has been an apparent discrepancy between measurements using long-range probes, such as fluorescence resonance energy transfer, and short-range probes, such as Trp-Cys quenching. In this work, we make both such measurements on the same protein, α-synuclein, and confirm that such discrepancy exists. Molecular dynamics simulations suggest that such differences result from a diffusion coefficient that depends on the spatial distance between probes. Diffusional estimates in good quantitative agreement with experiment are obtained by accounting for the distinct distance ranges probed by fluorescence resonance energy transfer and Trp-Cys quenching.