A general two-process model describes the hydrogen exchange behavior of RNase A in unfolding conditions.

When NMR hydrogen exchange was used previously to monitor the kinetics of RNase A unfolding, some peptide NH protons were found to show EX2 exchange (detected by base catalysis) in addition to the expected EX1 exchange, whose rate is limited by the kinetic unfolding process. In earlier work, two groups showed independently that a restricted two-process model successfully fits published hydrogen exchange rates of native RNase A in the range 0-0.7 M guanidinium chloride. We find that this model predicts properties that are very different from the observed properties of the EX2 exchange reactions of RNase A in conditions where guanidine-induced unfolding takes place. The model predicts that EX2 exchange should be too fast to measure by the technique used, whereas it is readily measurable. Possible explanations for the contradiction are considered here, and we show that removing the restriction from the earlier two-process model is sufficient to resolve the contradiction; instead of specifying that exchange caused by global unfolding occurs by the EX2 mechanism, we allow it to occur by the general mechanism, which includes both the EX1 and EX2 cases. It is logical to remove this restriction because global unfolding of RNase A is known to give rise to EX1 exchange in these unfolding conditions. Resolving the contradiction makes it possible to determine whether populated unfolding intermediates contribute to the EX2 exchange, and this question is considered elsewhere. The results and simulations indicate that moderate or high denaturant concentrations readily give rise to EX1 exchange in native proteins. Earlier studies showed that hydrogen exchange in native proteins typically occurs by the EX2 mechanism but that high temperatures or pH values above 7 may give rise to EX1 exchange. High denaturant concentrations should be added to the list of variables likely to cause EX1 exchange.

Mutant G protein α subunit activated by Gβγ: A model for receptor activation?

How receptors catalyze exchange of GTP for GDP bound to the Gα subunit of trimeric G proteins is not known. One proposal is that the receptor uses the G protein's βγ heterodimer as a lever, tilting it to pull open the guanine nucleotide binding pocket of Gα. To test this possibility, we designed a mutant Gα that would bind to βγ in the tilted conformation. To do so, we excised a helical turn (four residues) from the N-terminal region of αs, the α subunit of GS, the stimulatory regulator of adenylyl cyclase. In the presence, but not in the absence, of transiently expressed β1 and γ2, this mutant (αsΔ), markedly stimulated cAMP accumulation. This effect depended on the ability of the coexpressed β protein to interact normally with the lip of the nucleotide binding pocket of αsΔ. We substituted alanine for an aspartate in β1 that binds to a lysine (K206) in the lip of the α subunit's nucleotide binding pocket. Coexpressed with αsΔ and γ2, this mutant, β1-D228A, elevated cAMP much less than did β1-wild type; it did bind to αsΔ normally, however, as indicated by its unimpaired ability to target αsΔ to the plasma membrane. We conclude that βγ can activate αs and that this effect probably involves both a tilt of βγ relative to αs and interaction of β with the lip of the nucleotide binding pocket. We speculate that receptors use a similar mechanism to activate trimeric G proteins.

Rapid activation of Na+/H+ exchange by aldosterone in renal epithelial cells requires Ca2+ and stimulation of a plasma membrane proton conductance.

There is increasing evidence for an additional acute, nongenomic action of the mineralocorticoid hormone aldosterone on renal epithelial cells, leading to a two-step model of mineralocorticoid action on electrolyte excretion. We investigated the acute effect of aldosterone on intracellular free Ca2+ and on intracellular pH in an aldosterone-sensitive Madin-Darby canine kidney cell clone. Within seconds of application of aldosterone, but not of the glucocorticoid hydrocortisone, there was a 3-fold sustained increase of intracellular Ca2+ at a half-maximal concentration of 10(-10) mol/liter. Omission of extracellular Ca2+ prevented this hormone response. In the presence of extracellular Ca2+ aldosterone led to intracellular alkalinization. The Na+/H+ exchange inhibitor ethyl-isopropanol-amiloride (EIPA) prevented the aldosterone-induced alkalinization but not the aldosterone-induced increase of intracellular Ca2+. Omission of extracellular Ca2+ also prevented aldosterone-induced alkalinization. Instead, aldosterone led to a Zn(2+)-dependent intracellular acidification in the presence of EIPA, indicative of an increase of plasma membrane proton conductance. Under control conditions, Zn2+ prevented the aldosterone-induced alkalinization completely. We conclude that aldosterone stimulated net-entry of Ca2+ from the extracellular compartment and a plasma membrane H+ conductance as prerequisites for the stimulation of plasma membrane Na+/H+ exchange which in turn modulates K+ channel acitivity. It is probable that the aldosterone-sensitive H+ conductance maintains Na+/H+ exchange activity by providing an acidic environment in the vicinity of the exchanger. Thus, genomic action of aldosterone determines cellular transport equipment, whereas the nongenomic action regulates transporter activity that requires responses within seconds or minutes, which explains the rapid effects on electrolyte excretion.

Role of hydrophobic interactions and desolvation in determining the structural properties of a model alpha beta peptide.

Model AB, a 20-amino acid peptide that was designed to adopt an alpha beta tertiary structure stabilized by hydrophobic interactions between residues in adjacent helical and extended segments, exhibited large pKa shifts of several ionizable groups and slow hydrogen/deuterium exchange rates of nearly all the peptide amide groups [Butcher, D. J., Bruch, M. D. & Moe, G. T. (1995) Biopolymers 36, 109-120]. These properties, which depend on structure and hydration, are commonly observed in larger proteins but are quite unusual for small peptides. To identify which of several possible features of the peptide design are most important in determining these properties, several closely related analogs of Model AB were characterized by CD and NMR spectroscopy. The results show that hydrophobic interactions between adjacent helical and extended segments are structure-determining and have the additional effect of altering water-peptide interactions over much of the peptide surface. These results may have important implications for understanding mechanisms of protein folding and for the design of independently folding peptides.