Conformational Plasticity of proNGF

Nerve Growth Factor is an essential protein that supports neuronal survival during development and influences neuronal function throughout adulthood, both in the central and peripheral nervous system. The unprocessed precursor of NGF, proNGF, seems to be endowed with biological functions distinct from those of the mature protein, such as chaperone-like activities and apoptotic and/or neurotrophic properties. We have previously suggested, based on Small Angle X-ray Scattering data, that recombinant murine proNGF has features typical of an intrinsically unfolded protein. Using complementary biophysical techniques, we show here new evidence that clarifies and widens this hypothesis through a detailed comparison of the structural properties of NGF and proNGF. Our data provide direct information about the dynamic properties of the pro-peptide and indicate that proNGF assumes in solution a compact globular conformation. The N-terminal pro-peptide extension influences the chemical environment of the mature protein and protects the protein from proteolytic digestion. Accordingly, we observe that unfolding of proNGF involves a two-steps mechanism. The distinct structural properties of proNGF as compared to NGF agree with and rationalise a different functional role of the precursor

Isothermal Microcalorimetry to Investigate Non Specific Interactions in Biophysical Chemistry

Isothermal titration microcalorimetry (ITC) is mostly used to investigate the thermodynamics of “specific” host-guest interactions in biology as well as in supramolecular chemistry. The aim of this review is to demonstrate that ITC can also provide useful information about non-specific interactions, like electrostatic or hydrophobic interactions. More attention will be given in the use of ITC to investigate polyelectrolyte-polyelectrolyte (in particular DNA-polycation), polyelectrolyte-protein as well as protein-lipid interactions. We will emphasize that in most cases these “non specific” interactions, as their definition will indicate, are favoured or even driven by an increase in the entropy of the system. The origin of this entropy increase will be discussed for some particular systems. We will also show that in many cases entropy-enthalpy compensation phenomena occur

Protein intercalated bentonite recovered using adsorption from stickwater

Wastewater from the meat rendering industry is called stickwater and contains about 4 wt% protein and has a high biological oxygen demand (approximately 150,000 mg/l). Large volumes require treatment prior to disposal, leading to a potentially useful source of protein being lost. In this study the organic fraction of stickwater (largely protein) was recovered by adsorption onto bentonite followed by centrifugation. Adsorption behavior was examined for a range of pH and clay concentrations. Equilibrium behaviour was described using the Langmuir–Freundlich isotherm. 25% organics can be recovered from stickwater without clay at pH below 5. Organic matter recovery was between 75 and 90% at 1 g clay per 20 g solution. Recovery decreased with increasing pH above pH 7–25% at pH 12, but was independent of pH at higher clay concentrations. Bentonite basal spacing increased from 13 to 23.5 Å for all pHs showing that protein had intercalated between clay layers. At high pH, bentonite had a positive charge on its edges, giving an expanded structure with a low pellet density at 17% solids, whereas at pH below 5 pellet density was between 25 and 33% solids. This has implications for downstream processing because more water would need to be removed, increasing downstream processing costs. Using between 5 and 10 g clay/200 g solution, adsorption was sufficient to give greater than 60% organics recovery. Processing would therefore be a trade off between using as as little as possible clay to minimise cost and maximise percentage organics adsorbed per gram of clay, while maximising recovery and maintaining small process volumes