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A new method is described for direct monitoring of the conformational stability of proteins that are physically adsorbed from solution onto a solid substrate. The adsorption-induced conformational changes of insulin are studied using a combination of hydrogen/deuterium (H/D) exchange and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The effect of the surface hydrophobicity on the adsorption-induced conformational changes in the insulin structure is probed by adsorbing insulin on a hydrophilic silica and a hydrophobic methylated silica surface before subjecting the insulin molecules to the isotopic exchange process. The present study describes the experimental procedure of this new application of MALDI. Results show that insulin is more highly and more irreversibly adsorbed to a hydrophobic methylated silica surface than to a hydrophilic silica surface. Hydrogen-exchange experiments clearly demonstrate that the strong interaction of insulin with the hydrophobic surface is accompanied by a strong increase in the H/D-exchange rates and thus in a reduction in the insulin native structural stability. In contrast, H/D-exchange rates of insulin are somewhat reduced upon adsorption on silica from solution.

A flavodoxin from Azotobacter vinelandii is chosen as a model system to study the folding of α/ß doubly wound proteins. The guanidinium hydrochloride induced unfolding of apoflavodoxin is demonstrated to be reversible. Apoflavodoxin thus can fold in the absence of the FMN cofactor. The unfolding curves obtained for wild-type, C69A and C69S apoflavodoxin as monitored by circular dichroism and fluorescence spectroscopy do not coincide. Apoflavodoxin unfolding occurs therefore not via a simple two-state mechanism. The experimental data can be described by a three-state mechanism of apoflavodoxin equilibrium unfolding in which a relatively stable intermediate is involved. The intermediate species lacks the characteristic tertiary structure of native apoflavodoxin as deduced from fluorescence spectroscopy, but has significant secondary structure as inferred from circular dichroism spectroscopy. Both spectroscopic techniques show that thermally-induced unfolding of apoflavodoxin also proceeds through formation of a similar molten globule-like species. Thermal unfolding of apoflavodoxin is accompanied by anomalous circular dichroism characteristics: the negative ellipticity at 222 nM increases in the transition zone of unfolding. This effect is most likely attributable to changes in tertiary interactions of aromatic side chains upon protein unfolding. From the presented results and hydrogen/deuterium exchange data, a model for the equilibrium unfolding of apoflavodoxin is presented. [ABSTRACT FROM AUTHOR]

This study deals with the detailed electrochemistry and complete EPR-monitored titrations of flavodoxin II of Azotobacter vinelandii (ATCC 478). Since wild-type flavodoxin dimerises via intermolecular disulphide bond formation between Cys69 residues. Cys69 has been replaced by both an alanine and a serine residue. Redox properties of she C69A and C69S flavodoxin mutants were compared to those of wild-type flavodoxin. In the presence of the promotor neomycin, C69A and C69S flavodoxin showed a reversible response of the semiquinone/hydroquinone couple at the glassy carbon electrode. However, the addition of dithiothreitol proved to be necessary for the stabilisation of the wild-type flavodoxin response. EPR-monitored redox titrations of wild-type and C69A flavodoxin at high and low pH confirmed the redox potentials measured using cyclic voltammetry. The pH dependence of the semiquinone/hydroquinone redox potentials cannot be described using a model assuming one redox-linked pK. Instead, the presence of at least two redox-linked protonation sites is suggested: pKred,1 = 5.39 ± 0.08. pKox = 7.29 ± 0.14, and pKred,2 = 7.84 ± 0.14 with Em,7 = -459 ± 4 mV, and a constant redox potential at high pH of -485 ± mV. The dependence of the semiquinone/hydroquionone redox potential on temperature is -0.5 ± 0.1 mV · K-1, yielding ΔH° = 28.6 ± 1.5 kJ · mol-1 and ΔS° = -50.0 ± 6.2 J · mol -1 · K-1. No significant differences in redox properties of wild-type. C69A, and C69S flavodoxin were observed. The electrochemical data suggest that replacement of Cys69 in the vicinity of the FMN by either an alanine or a serine residue does not alter the dielectric properties and structure of A. vinelandii flavodoxin 11. [ABSTRACT FROM AUTHOR]

Antibodies of type IgG may be divided into two classes, called lambda or kappa, depending on the type of light chain. We have identified a conserved pocket between the two domains CH1 and CL of human IgG kappa-Fab, which is not present in the lambda type. This pocket was used as a target docking site with the purpose of exploring the possibilities of designing affinity ligands that could function as such even after immobilization to gel. The idea of the design arose mainly from the results of the saturated transfer difference (STD-NMR) screening of 46 compounds identified by means of virtual docking of 60 K diverse compounds from the Available Chemicals Directory (ACD). Surface plasmon resonance (SPR) was used as an alternative method to monitor binding in solution. A total of 24 compounds belonging to a directed library were designed, synthesized, and screened in solution. They consist essentially of an amino acid condensed to a N,N'-methylated phenyl urea. STD-NMR results suggest that a small hydrophobic side chain in the condensed amino acid promotes binding, whereas a hydroxyl-group-containing side chain implies absence of STD-NMR signals. Three compounds of the directed library were immobilized and evaluated as chromatographic probes. In one case, using D-Pro as the condensed amino acid, columns packed with ligand-coupled Sepharose (Amersham Biosciences) retained two different monoclonal samples of kappa-Fab fragments with different variable regions, whereas a sample of monoclonal lambda-Fab fragments was not retained under similar chromatographic conditions.