The aggregation of cytochrome C may be linked to its flexibility during refolding

Large-scale expression of biopharmaceutical proteins in cellular hosts results in production of large insoluble mass aggregates. In order to generate functional product, these aggregates require further processing through refolding with denaturant, a process in itself that can result in aggregation. Using a model folding protein, cytochrome C, we show how an increase in final denaturant concentration decreases the propensity of the protein to aggregate during refolding. Using polarised fluorescence anisotropy, we show how reduced levels of aggregation can be achieved by increasing the period of time the protein remains flexible during refolding, mediated through dilution ratios. This highlights the relationship between the flexibility of a protein and its propensity to aggregate. We attribute this behaviour to the preferential urea-residue interaction, over self-association between molecules

Comprehensive determination of 3JHNHα for unfolded proteins using 13C′-resolved spin-echo difference spectroscopy

An experiment is presented to determine 3JHNHα coupling constants, with significant advantages for applications to unfolded proteins. The determination of coupling constants for the peptide chain using 1D 1H, or 2D and 3D 1H-15N correlation spectroscopy is often hampered by extensive resonance overlap when dealing with flexible, disordered proteins. In the experiment detailed here, the overlap problem is largely circumvented by recording 1H-13C′ correlation spectra, which demonstrate superior resolution for unfolded proteins. J-coupling constants are extracted from the peak intensities in a pair of 2D spin-echo difference experiments, affording rapid acquisition of the coupling data. In an application to the cytoplasmic domain of human neuroligin-3 (hNlg3cyt) data were obtained for 78 residues, compared to 54 coupling constants obtained from a 3D HNHA experiment. The coupling constants suggest that hNlg3cyt is intrinsically disordered, with little propensity for structure

Mechanisms for the Evolution of a Derived Function in the Ancestral Glucocorticoid Receptor

Understanding the genetic, structural, and biophysical mechanisms that caused protein functions to evolve is a central goal of molecular evolutionary studies. Ancestral sequence reconstruction (ASR) offers an experimental approach to these questions. Here we use ASR to shed light on the earliest functions and evolution of the glucocorticoid receptor (GR), a steroid-activated transcription factor that plays a key role in the regulation of vertebrate physiology. Prior work showed that GR and its paralog, the mineralocorticoid receptor (MR), duplicated from a common ancestor roughly 450 million years ago; the ancestral functions were largely conserved in the MR lineage, but the functions of GRs—reduced sensitivity to all hormones and increased selectivity for glucocorticoids—are derived. Although the mechanisms for the evolution of glucocorticoid specificity have been identified, how reduced sensitivity evolved has not yet been studied. Here we report on the reconstruction of the deepest ancestor in the GR lineage (AncGR1) and demonstrate that GR's reduced sensitivity evolved before the acquisition of restricted hormone specificity, shortly after the GR–MR split. Using site-directed mutagenesis, X-ray crystallography, and computational analyses of protein stability to recapitulate and determine the effects of historical mutations, we show that AncGR1's reduced ligand sensitivity evolved primarily due to three key substitutions. Two large-effect mutations weakened hydrogen bonds and van der Waals interactions within the ancestral protein, reducing its stability. The degenerative effect of these two mutations is extremely strong, but a third permissive substitution, which has no apparent effect on function in the ancestral background and is likely to have occurred first, buffered the effects of the destabilizing mutations. Taken together, our results highlight the potentially creative role of substitutions that partially degrade protein structure and function and reinforce the importance of permissive mutations in protein evolution

Methods of limiting decomposition and Markovian summation in queueing system with infinite number of servers

In this paper, we study the process generated by the customers which are serviced in a queueing system with an infinite number of servers. Two methods referred to as the method of limiting decomposition and the method of Markovian summation are proposed, implemented and compared. The characteristic function of the probability distribution for the studied process is obtained. The numerical examples are performed for different values of queueing system characteristics

Incremental and Average Control Costs in a Model of Water Quality Trading with Discrete Abatement Units

Discrete abatement, Incremental control cost, Average control cost, Willingness to pay, D61, Q53,

New Techniques for the Construction of Residue Potentials for Protein Folding

. A smooth empirical potential is constructed for use in off-lattice protein folding studies. Our potential is a function of the amino acid labels and of the distances between the C ff atoms of a protein. The potential is a sum of smooth surface potential terms that model solvent interactions and of pair potentials that are functions of a distance, with a smooth cutoff at 12 Angstrøm. Techniques include the use of a fully automatic and reliable estimator for smooth densities, of cluster analysis to group together amino acid pairs with similar distance distributions, and of quadratic programming to find appropriate weights with which the various terms enter the total potential. For nine small test proteins, the new potential has minima within 1.3--4.7 A of the PDB geometry, with one exception that has an error of 8.5 A. Moreover, a nonuniqueness theorem is given that shows that no set of equilibrium geometries can determine the true effective potential energy function. Keywords. protei..