Exposure Of Thiol Groups Research Articles

Exposure of thiol groups in the heat-induced denaturation of β-lactoglobulin

Protein aggregates can be stabilised by disulphide bridges. The whey protein β-lactoglobulin (β-lac) contains a disulphide bridge and a free cysteine that are shielded from the solvent by an α-helix. These groups are important in the thiol–disulphide exchange that occurs during aggregation and gelation of β-lac. Replica exchange molecular dynamics simulations show that the exposure mechanism is very different for the two buried groups. While melting of the α-helix enhances exposure of the free cysteine, it does not for the buried bridge. These findings shed light on the molecular mechanism of the first step of β-lac denaturation and aggregation.

Evidence for the Regulatory Role of the N-terminal Helix of Secretory Phospholipase A2 from Studies on Native and Chimeric Proteins

The phospholipase A(2) (PLA(2)) enzymes are activated by binding to phospholipid membranes. Although the N-terminal alpha-helix of group I/II PLA(2)s plays an important role in the productive mode membrane binding of the enzymes, its role in the structural aspects of membrane-induced activation of PLA(2)s is not well understood. In order to elucidate membrane-induced conformational changes in the N-terminal helix and in the rest of the PLA(2), we have created semisynthetic human group IB PLA(2) in which the N-terminal decapeptide is joined with the (13)C-labeled fragment, as well as a chimeric protein containing the N-terminal decapeptide from human group IIA PLA(2) joined with a (13)C-labeled fragment of group IB PLA(2). Infrared spectral resolution of the unlabeled and (13)C-labeled segments suggests that the N-terminal helix of membrane-bound IB PLA(2) has a more rigid structure than the other helices. On the other hand, the overall structure of the chimeric PLA(2) is more rigid than that of the IB PLA(2), but the N-terminal helix is more flexible. A combination of homology modeling and polarized infrared spectroscopy provides the structure of membrane-bound chimeric PLA(2), which demonstrates remarkable similarity but also distinct differences compared with that of IB PLA(2). Correlation is delineated between structural and membrane binding properties of PLA(2)s and their N-terminal helices. Altogether, the data provide evidence that the N-terminal helix of group I/II PLA(2)s acts as a regulatory domain that mediates interfacial activation of these enzymes.

Nativelike intermediate on the unfolding pathway of pig kidney fructose-1,6-bisphosphatase.

The unfolding and dissociation of the tetrameric enzyme fructose-1,6-bisphosphatase from pig kidney by guanidine hydrochloride have been investigated at equilibrium by monitoring enzyme activity, ANS binding, intrinsic (tyrosine) protein fluorescence, exposure of thiol groups, fluorescence of extrinsic probes (AEDANS, MIANS), and size-exclusion chromatography. The unfolding is a multistate process involving as the first intermediate a catalytically inactive tetramer. The evidence that indicates the existence of this intermediate is as follows: (1) the loss of enzymatic activity and the concomitant increase of ANS binding, at low concentrations of Gdn.HCl (midpoint at 0.75 M), are both protein concentration independent, and (2) the enzyme remains in a tetrameric state at 0.9 M Gdn.HCl as shown by size-exclusion chromatography. At slightly higher Gdn.HCl concentrations the inactive tetramer dissociates to a compact dimer which is prone to aggregate. Further evidence for dissociation of tetramers to dimers and of dimers to monomers comes from the concentration dependence of AEDANS-labeled enzyme anisotropy data. Above 2.3 M Gdn.HCl the change of AEDANS anisotropy is concentration independent, indicative of monomer unfolding, which also is detected by a red shift of MIANS-labeled enzyme emission. At Gdn.HCl concentrations higher than 3.0 M, the protein elutes from the size-exclusion column as a single peak, with a retention volume smaller than that of the native protein, corresponding to the completely unfolded monomer. In the presence of its cofactor Mg(2+), the denaturated enzyme could be successfully reconstituted into the active enzyme with a yield of approximately 70-90%. Refolding kinetic data indicate that rapid refolding and reassociation of the monomers into a nativelike tetramer and reactivation of the tetramer are sequential events, the latter involving slow and small conformational rearrangements in the refolded enzyme.

Inactivation, subunit dissociation, aggregation, and unfolding of myosin subfragment 1 during guanidine denaturation.

The effect of guanidine hydrochloride on ATPase activity, gel filtration, turbidity, exposure of thiol groups, far-UV circular dichroism, and the fluorescence emission intensity of myosin subfragment 1 (S-1) was studied under equilibrium conditions. It was found that the denaturation process involves several intermediate states. The enzymatic activity of S-1 is at first lost at very low concentrations of GdnHCl (lower than 0.5 M). At a slightly higher GdnHCl concentration (about 0.5 M), the light chains dissociate and this dissociation is closely followed by the formation of aggregates between the naked heavy chains of S-1 molecules in the guanidine hydrochloride range of concentrations 0.5-1 M. At GdnHCl concentrations above 1 M, aggregates gradually disappear and S-1 loses its secondary and tertiary structures. These phenomena are partly reversible, and ATPase activity is only partially recovered under highly limited conditions. These results are discussed in relation to the nature of myosin subunit assembly. The head fragment of 20 kDa is thus suggested to be implicated in the binding of light chain to heavy chain and in the self-association of free heavy chains.

Evidence for essential thiol groups and disulfide bonds in agonist and antagonist binding to the dopamine receptor

Dithiothreitol (DTT), a disulfide reducing agent, diminished the specific binding of [ 3H] dopamine to partially purified calf striatal membranes (P 2) but did not have an effect on [ 3H] spiroperidol binding. The thiol reagents, p-chloromercuribenzoate (PCMB), N-ethylmaleimide (NEM) and iodoacetamide (IA), were also tested for inhibitory effects on agonist and antagonist binding to the dopamine receptor. PCMB inhibited both [ 3H] dopamine and [ 3H] spiroperidol binding by changing the affinity (K d) and the number of binding sites (B max) for both of these ligands. This effect of PCMB was reversed by the addition of DTT. NEM inhibited binding to the dopamine agonist site but not to the antagonist site, while IA was ineffective on either site. These results indicate that a DTT-reducible disulfide bond may be an essential component for agonist binding to the dopamine receptor. Furthermore, the experiments with PCMB, NEM and IA suggest that the exposure of thiol groups in the dopamine receptor may play an important role in agonist and antagonist binding.