title: Stability of tethered proteins.
abstract: The stability of tethered globular proteins under denaturing conditions was interrogated with a hydrophobic surface, since conventional structural methods like circular dichroism (CD) and fluorescence or infrared spectroscopy could not be used because of the presence of an opaque solid substrate and extremely low surface concentrations. For free protein in solution, CD spectra gave well-known unfolding denaturing curves for lysozyme (LYS) and ribonuclease A (RNase A). The unfolding process for covalently tethered LYS and RNase A was followed, with multimolecular force spectroscopy (using an atomic force microscope in force-mode), via the adhesion energy between a functionalized self-assembled monolayer (CH(3)-SAM) probe and the protein molecules covalently bound to a carboxylic SAM on a gold-coated glass coverslip. The adhesion energy passed through a maximum for the tethered proteins during excursions with temperature or chemical denaturants. The initial rise in adhesion energy on increasing the temperature or GuHCl concentration was due to increasing exposure of the unfolded hydrophobic core of the proteins to the CH(3)-SAM tip, while the decrease in adhesion energy at high temperature or large concentrations of denaturant is attributed to interprotein association with nearest neighbors. Attempts to recover their folded state upon cooling (or reducing GuHCl concentration) were unsuccessful. Also, dilution of surface-tethered LYS reduced the aggregation with nearest neighbors about 6-fold. These results are in qualitative agreement with Monte Carlo simulations on a simple two-letter lattice protein model, especially for low concentrations of grafted proteins.
authors.affiliation: Howard P. Isermann Department of Chemical and Biological Engineering and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180-3590, USA.
authors.forename: Gaurav
authors.initials: G
authors.lastname: Anand
authors.forename: Sumit
authors.initials: S
authors.lastname: Sharma
authors.forename: Sanat K
authors.initials: SK
authors.lastname: Kumar
authors.forename: Georges
authors.initials: G
authors.lastname: Belfort