IgG rheumatoid factors against the four human Fc-gamma subclasses in early rheumatoid arthritis (the Swedish TIRA project).

Rheumatoid factor (RF), i.e. a family of autoantibodies against the Fc part of IgG, is an important seromarker of rheumatoid arthritis (RA). Traditional particle agglutination without disclosing the antibody isotype remains the predominating diagnostic method in clinical routine. Although IgG-RF attracts pathogenic interest, its detection remains technically challenging. The present study aimed at developing a set of tests identifying IgG-RFs directed against the four IgG subclasses. IgG-RF against either subclass of human IgG-Fc were analysed with four novel enzyme-linked immunosorbent assays (ELISAs) utilizing four recombinant human Fc-gamma fragments (hIgG1-4) as sources of antigen. Sera from 40 patients with recent onset RA (20 seropositive and 20 seronegative by IgM-RF and IgA-RF-isotype-specific ELISA) were analysed. Sera from 20 healthy blood donors served as reference. Among the IgM-/IgA-RF-positive RA-sera, IgG-RF was found directed against hIgG1 and hIgG2, but not against hIgG3 or hIgG4. Significant correlations were seen between IgG-RF against hIgG2-Fc and IgM-RF (r = 0.666) levels. Further prospective studies are warranted to elucidate any correlation to disease course and outcome.

Synthetic de novo designed polypeptides for control of nanoparticle assembly and biosensing.

This contribution describes how de novo designed synthetic helix-loop-helix polypeptides are utilized to control the assembly of gold nanoparticles and as scaffolds for biosensing. The synthetic polypeptides are designed to fold into a four-helix bundle upon dimerization. When immobilized on gold nanoparticles, dimerization and folding occur between peptides on neighbouring particles as an effect of particle aggregation and the folded polypeptides are rigid enough to keep the particles separated at a distance corresponding to the size of the four-helix bundle. Moreover, peptide dimerization offers a convenient route to assemble nanoparticles into hybrid multilayers on planar substrates. The drastic change in the resonance conditions of the localized nanoparticle surface plasmon upon particle aggregation is shown to be useful for optical detection of biomolecular interactions.

Alpha-helix-inducing dimerization of synthetic polypeptide scaffolds on gold.

Designed, synthetic polypeptides that assemble into four-helix bundles upon dimerization in solution were studied with respect to folding on planar gold surfaces. A model system with controllable dimerization properties was employed, consisting of negatively and positively charged peptides. Circular dichroism spectroscopy and surface plasmon resonance based measurements showed that at neutral pH, the peptides were able to form heterodimers in solution, but unfavorable electrostatic interactions prevented the formation of homodimers. The dimerization propensity was found to be both pH- and buffer-dependent. A series of infrared absorption-reflection spectroscopy experiments of the polypeptides attached to planar gold surfaces revealed that if the negatively charged peptide was immobilized from a loading solution where it was folded, its structure was retained on the surface provided it had a cysteine residue available for anchoring to gold. If it was immobilized as random coil, it remained unstructured on the surface but was able to fold through heterodimerization if subsequently exposed to a positively charged polypeptide. When the positively charged peptide was immobilized as random coil, heterodimerization could not be induced, probably because of high-affinity interactions between the charged primary amine groups and the gold surface. These observations are intended to pave the way for future engineering of functional surfaces based on polypeptide scaffolds where folding is known to be crucial for function.

Subtle differences in dissociation rates of interactions between destabilized human carbonic anhydrase II mutants and immobilized benzenesulfonamide inhibitors probed by a surface plasmon resonance biosensor.

The development of commercial biosensors based on surface plasmon resonance has made possible careful characterization of biomolecular interactions. Here, a set of destabilized human carbonic anhydrase II (HCA II) mutants was investigated with respect to their interaction kinetics with two different immobilized benzenesulfonamide inhibitors. Point mutations were located distantly from the active site, and the destabilization energies were up to 23 kJ/mol. The dissociation rate of wild-type HCA II, as determined from the binding to the inhibitor with higher affinity, was 0.019 s(-1). For the mutants, dissociation rates were faster (0.022-0.025 s(-1)), and a correlation between faster dissociation and a high degree of destabilization was observed. We interpreted these results in terms of increased dynamics of the tertiary structures of the mutants. This interpretation was supported by entropy determinations, showing that the entropy of the native structure significantly increased upon destabilization of the protein molecule. Our findings demonstrate the applicability of modern biosensor technology in the study of subtle details in molecular interaction mechanisms, such as the long-range effect of point mutations on interaction kinetics.

Energy-linked nicotinamide nucleotide transhydrogenase. Properties of proton-translocating mitochondrial transhydrogenase from beef heart purified by fast protein liquid chromatography.

Mitochondrial nicotinamide nucleotide transhydrogenase from beef heart was purified by a novel procedure involving fast protein liquid chromatography and characterized with respect to molecular and catalytic properties. The method is reproducible, gives highly pure transhydrogenase as judged by silver staining, and can be modified to produce large amounts of pure transhydrogenase protein suitable for e.g. sequencing and other protein chemical studies. Transhydrogenase purified by fast protein liquid chromatography is reconstitutively active and pumps protons as indicated by an extensive quenching of 9-aminoacridine fluorescence. Under conditions which generate a proton gradient in the absence of a membrane potential the activity of reconstituted transhydrogenase is close to zero indicating a complete and proper incorporation in the membrane and a preferential regulation of the enzyme by a proton gradient rather than a membrane potential. Treatment of reconstituted transhydrogenase with N,N-dicyclohexylcarbodiimide results in an inhibition of proton pump activity without an effect on uncoupled catalytic activity, suggesting that proton translocation and catalytic activities are not obligatory linked or that this agent separates proton pumping from the catalytic activity.

Energy-linked nicotinamide nucleotide transhydrogenase. Kinetics and regulation of purified and reconstituted transhydrogenase from beef heart mitochondria.

1. Purified and reconstituted nicotinamide nucleotide transhydrogenase from beef heart mitochondria was investigated with respect to kinetic and regulatory properties in uncoupled and coupled liposomes. 2. Double reciprocal plots of initial velocities for the reduction of NAD+ by NADPH versus substrate concentrations were convergent and intersecting on or close to the abscissa, indicating a ternary complex mechanism. The effect of site-specific inhibitors indicates that the order of addition of the substrates to the enzyme is random. 3. Reconstituted transhydrogenase uncoupled by FCCP reveals kinetic properties that are indicative of energization, i.e. an increased and decreased affinity for NADP+ and NAD+, respectively, suggesting that reconstituted transhydrogenase is maintained in an activated conformation. An increased extent of coupling causes a progressively increasing change in the same direction. These results suggest that the uncoupler-dependent enhancement of the rate of reduction of NAD+ by NADPH is due to a decreased Km for NAD+. 4. Reconstituted transhydrogenase catalyzes a transhydrogenation between NADH and 3-acetylpyridine adenine dinucleotide (oxidized) in the presence of NADPH. Reconstituted transhydrogenase also also catalyzes the reduction of thio-NADP+ by NADPH in the presence of NADH. Both reactions are concluded to occur indirectly through the generation of NADP+ and NAD+, respectively, and not directly through a reduced enzyme intermediate. 5. A proton pump mechanism is proposed for transhydrogenase which involves a dimeric form of the enzyme where the two subunits are alternating in proton pumping.