PMID: 15001361;Abstract:
The four-helical immunity protein Im7 folds through an on-pathway intermediate that has a specific, but partially misfolded, hydrophobic core. In order to gain further insight into the structure of this species, we have identified the backbone hydrogen bonds formed in the ensemble by measuring the amide exchange rates (under EX2 conditions) of the wild-type protein and a variant, I72V. In this mutant the intermediate is significantly destabilised relative to the unfolded state (ΔΔGui=4.4kJ/mol), but the native state is only slightly destabilised (ΔΔG nu=1.8kJ/mol) at 10°C in 2H2O, pH* 7.0 containing 0.4 M Na2SO4, consistent with the view that this residue forms significant non-native stabilising interactions in the intermediate state. Comparison of the hydrogen exchange rates of the two proteins, therefore, enables the state from which hydrogen exchange occurs to be identified. The data show that amides in helices I, II and IV in both proteins exchange slowly with a free energy similar to that associated with global unfolding, suggesting that these helices form highly protected hydrogen-bonded helical structure in the intermediate. By contrast, amides in helix III exchange rapidly in both proteins. Importantly, the rate of exchange of amides in helix III are slowed substantially in the Im7* variant, I72V, compared with the wild-type protein, whilst other amides exchange more rapidly in the mutant protein, in accord with the kinetics of folding/unfolding measured using chevron analysis. These data demonstrate, therefore, that local fluctuations do not dominate the exchange mechanism and confirm that helix III does not form stable secondary structure in the intermediate. By combining these results with previously obtained Φ-values, we show that the on-pathway folding intermediate of Im7 contains extensive, stable hydrogen-bonded structure in helices I, II and IV, and that this structure is stabilised by both native and non-native interactions involving amino acid side-chains in these helices. © 2004 Elsevier Ltd. All rights reserved.
TOR kinase complex I (TORC1) is a key regulator of cell growth and metabolism in all eukaryotes. Previous studies in yeast have shown that three GTPases-Gtr1, Gtr2, and Rho1-bind to TORC1 in nitrogen and amino acid starvation conditions to block phosphorylation of the S6 kinase Sch9 and activate protein phosphatase 2A (PP2A). This leads to downregulation of 450 Sch9-dependent protein and ribosome synthesis genes and upregulation of 100 PP2A-dependent nitrogen assimilation and amino acid synthesis genes. Here, using bandshift assays and microarray measurements, we show that the TORC1 pathway also populates three other stress/starvation states. First, in glucose starvation conditions, the AMP-activated protein kinase (AMPK/Snf1) and at least one other factor push the TORC1 pathway into an off state, in which Sch9-branch signaling and PP2A-branch signaling are both inhibited. Remarkably, the TORC1 pathway remains in the glucose starvation (PP2A inhibited) state even when cells are simultaneously starved for nitrogen and glucose. Second, in osmotic stress, the MAPK Hog1/p38 drives the TORC1 pathway into a different state, in which Sch9 signaling and PP2A-branch signaling are inhibited, but PP2A-branch signaling can still be activated by nitrogen starvation. Third, in oxidative stress and heat stress, TORC1-Sch9 signaling is blocked while weak PP2A-branch signaling occurs. Together, our data show that the TORC1 pathway acts as an information-processing hub, activating different genes in different conditions to ensure that available energy is allocated to drive growth, amino acid synthesis, or a stress response, depending on the needs of the cell.
PMID: 23643537;PMCID: PMC3672359;Abstract:
Cells respond to stress and starvation by adjusting their growth rate and enacting stress defense programs. In eukaryotes this involves inactivation of TORC1, which in turn triggers downregulation of ribosome and protein synthesis genes and upregulation of stress response genes. Here we report that the highly conserved inositol pyrophosphate (PP-IP) second messengers (including 1-PP-IP5, 5-PP-IP4, and 5-PP-IP5) are also critical regulators of cell growth and the general stress response, acting in parallel with the TORC1 pathway to control the activity of the class I histone deacetylase Rpd3L. In fact, yeast cells that cannot synthesize any of the PP-IPs mount little to no transcriptional response to osmotic, heat, or oxidative stress. Furthermore, PP-IP-dependent regulation of Rpd3L occurs independently of the role individual PP-IPs (such as 5-PP-IP5) play in activating specialized stress/starvation response pathways. Thus, the PP-IP second messengers simultaneously activate and tune the global response to stress and starvation signals. © 2013 The Authors.
PMID: 9519300;Abstract:
New studies have shown that folding of β-sheet proteins can occur with and without intermediates, with fast to slow refolding rates and late to very late transition states. These experiments demonstrate that, despite early speculation to the contrary, β-sheet protein folding does not appear to be fundamentally different from that of helical and mixed α,β proteins.
