Solid-state synthesis and mechanical unfolding of polymers of T4 lysozyme.

Recent advances in single molecule manipulation methods offer a novel approach to investigating the protein folding problem. These studies usually are done on molecules that are naturally organized as linear arrays of globular domains. To extend these techniques to study proteins that normally exist as monomers, we have developed a method of synthesizing polymers of protein molecules in the solid state. By introducing cysteines at locations where bacteriophage T4 lysozyme molecules contact each other in a crystal and taking advantage of the alignment provided by the lattice, we have obtained polymers of defined polarity up to 25 molecules long that retain enzymatic activity. These polymers then were manipulated mechanically by using a modified scanning force microscope to characterize the force-induced reversible unfolding of the individual lysozyme molecules. This approach should be general and adaptable to many other proteins with known crystal structures. For T4 lysozyme, the force required to unfold the monomers was 64 +/- 16 pN at the pulling speed used. Refolding occurred within 1 sec of relaxation with an efficiency close to 100%. Analysis of the force versus extension curves suggests that the mechanical unfolding transition follows a two-state model. The unfolding forces determined in 1 M guanidine hydrochloride indicate that in these conditions the activation barrier for unfolding is reduced by 2 kcal/mol.

Calcium-dependent inactivation of L-type calcium channels in planar lipid bilayers.

Intracellular Ca2+ can inhibit the activity of voltage-gated Ca channels by modulating the rate of channel inactivation. Ca(2+)-dependent inactivation of these channels may be a common negative feedback process important for regulating Ca2+ entry under physiological and pathological conditions. This article demonstrates that the inactivation of cardiac L-type Ca channels, reconstituted into planar lipid bilayers and studied in the presence of a dihydropyridine agonist, is sensitive to Ca2+. The rates and extents of inactivation, determined from ensemble averages of unitary Ba2+ currents, decreased when the calcium concentration facing the intracellular surface of the channel ([Ca2+]i) was lowered from approximately 10 microM to 20 nM by the addition of Ca2+ chelators. The rates and extents of Ba2+ current inactivation could also be increased by subsequent addition of Ca2+ raising the [Ca2+]i to 15 microM, thus demonstrating that the Ca2+ dependence of inactivation could be reversibly regulated by changes in [Ca2+]i. In addition, reconstituted Ca channels inactivated more quickly when the inward current was carried by Ca2+ than when it was carried by Ba2+, suggesting that local increases in [Ca2+]i could activate Ca(2+)-dependent inactivation. These data support models in which Ca2+ binds to the channel itself or to closely associated regulatory proteins to control the rate of channel inactivation, and are inconsistent with purely enzymatic models for channel inactivation.

Conantokin-G antagonism of the NMDA receptor subtype expressed in cultured cerebellar granule cells.

Conantokins are peptide antagonists of the N-methyl-D-aspartate (NMDA) subclass of excitatory amino acid receptors. We compared conantokin-G and AP5 antagonism of NMDA receptor activity expressed in cultures of neonatal rat cerebellar granule cells, using the fluorescent calcium indicator dye fura-2. The results were consistent with the binding of two molecules of agonist (NMDA) for channel activation and one antagonist molecule (AP5) for inhibition. However, conantokin-G antagonism was more complex: the peptide inhibited only approximately 70% of the elevation of intracellular free calcium produced by NMDA. These results, when combined with previous ones [8], suggest that conantokin-G may have different affinities for, and functional effects on, different subtypes of NMDA receptor complexes expressed in the mammalian CNS.

A new Conus peptide ligand for Ca channel subtypes.

A cDNA clone encoding a new omega-conotoxin was identified from Conus magus. The predicted peptide was chemically synthesized using a novel strategy that efficiently yielded the biologically active disulfide-bonded isomer. This peptide, omega-conotoxin MVIID, targets other voltage-gated calcium channels besides the N-subtype and exhibits greater discrimination against the N-channel subtype than any other omega-conotoxin variant to date. Consequently, omega-conotoxin MVIID may be a particularly useful ligand for calcium channel subtypes that are not of the L- or N-subclasses. Of the eight major sequence variants of omega-conotoxins that have been elucidated, four come from Conus magus venom. We suggest that sequence variants from the same venom may be designed to optimally interact with different molecular variants of calcium channels; such omega-conotoxin sets from a single venom may therefore be useful for helping to identify novel calcium channel subtypes.

Biotinylated derivatives of omega-conotoxins GVIA and MVIID: probes for neuronal calcium channels.

The omega-conotoxins are small, disulfide-rich peptides which inhibit voltage-sensitive calcium channels. Biotinylated omega-conotoxins are potentially useful reagents for characterizing distinct subsets of calcium channels. We describe the preparation and characterization of biotinylated derivatives of two specific omega-conotoxins, GVIA and MVIID, which bind different calcium channel subtypes. Eight biotinylated derivatives were tested; all specifically displaced binding of the radiolabeled unbiotinylated omega-conotoxin. In general, the addition of one biotin moiety decreased the apparent affinity for the receptor target site by only approximately 10-fold. However, derivatization of omega-conotoxin MVIID at the Lys10 residue caused a much more marked effect, a ca 500-fold decrease in affinity. These results indicate that the vicinity of the Lys10 residue of omega-conotoxin MVIID may be more critical for binding to the receptor target site than regions around other amino groups in omega-conotoxins GVIA and MVIID. Thus, high affinity biotinylated omega-conotoxin GVIA and MVIID derivatives have been chemically defined; the biotin groups have been shown to be accessible to streptavidin. Given the commercial availability of streptavidin coupled to various reporter groups, the biotinylated omega-conotoxin derivatives described here should be widely useful for fluorescence, electron microscopic or immunological applications.

Conantokin-T. A gamma-carboxyglutamate containing peptide with N-methyl-d-aspartate antagonist activity.

Conantokin-T, a 21-amino acid peptide which induces sleep-like symptoms in young mice was purified from the venom of the fish-hunting cone snail, Conus tulipa. The amino acid sequence of the peptide was determined and verified by chemical synthesis. The peptide has 4 residues of the modified amino acid, gamma-carboxyglutamate (Gla). The sequence of the peptide is: Gly-Glu-Gla-Gla-Tyr-Gln-Lys-Met-Leu-Gla-Asn-Leu-Arg-Gla-Ala-Glu-Val-Lys- Lys-Asn-Ala-NH2. Conantokin-T inhibits N-methyl-D-aspartate (NMDA) receptor-mediated calcium influx in central nervous system neurons. This observation suggests that like conantokin-G (a homologous Conus peptide with recently identified NMDA antagonist activity) conantokin-T has NMDA antagonist activity. A sequence comparison of conantokins-T and -G identifies the 4 Gla residues and the N-terminal dipeptide sequence as potential key elements for the biological activity of this peptide.

Mutational analysis of a protein-folding pathway.

The effects of amino-acid replacements on the disulphide-coupled folding pathway of bovine pancreatic trypsin inhibitor have been examined. Replacements at three sites destabilize the native protein relative to the unfolded state, but have different effects on the relative stabilities of the disulphide-bonded folding intermediates, thus allowing the roles of the altered residues during folding to be distinguished.