Protein unfolding can be induced both by heating and by cooling from ambient temperatures. 1 Accurate analysis of heat and cold denaturation processes has the potential to unveil hitherto obscure aspects of protein stability and dynamics. 2 For instance, while heat denaturation is generally highly cooperative, cold denaturation has been suggested to occur in a noncooperative fashion. 3,4 This view has been recently supported by an NMR study of ubiquitin in reverse micelles at very low temperatures, 5 but this is still controversial since Van Horn et al., 6 on the basis of similar NMR data, and Kitahara et al., 7 by an NMR study at 2 kbar, found a simple two-state behavior for the low-temperature unfolding of ubiquitin.To reach a consensus on this debate and other general issues, it is necessary to investigate cold denaturation further. However, since the cold denaturation of most proteins occurs well below the freezing point of water, full access to the cold denatured state is normally limited for the obvious reason that water freezes at 0 °C. The most common approach to circumvent this difficulty has been to try to raise the temperature of cold denaturation using destabilizing agents such as extreme pH values, chemical denaturants, cryosolvents, or very high pressure. 7-10 Alternatively, some laboratories used proteins destabilized by a combination of point mutations and denaturing agents. 9 The main drawback of these approaches is that it is not generally easy to extrapolate results to physiological conditions. On the other hand, there are methods aimed at keeping water in a supercooled condition, but these studies have also invariably used destabilized proteins. 11,12Following a different approach, we looked for a protein whose cold denaturation could be studied without the need for destabilization in a normal buffer at physiological pH within a temperature range accessible to several techniques. Here we describe the cold and heat denaturation of yeast frataxin (Yfh1) measured both by NMR and CD spectroscopies. In a systematic study of the factors that influence the thermal stability of the frataxin fold, we had previously shown that although they share the same fold, three orthologues from E. coli (CyaY), S. cerevisiae (Yfh1) and H. sapiens (hfra), are characterized, under the same conditions, by a remarkable variation of melting temperatures. 13 Yfh1, the one with lowest heat denaturation temperature, seemed a promising candidate for cold denaturation above 0 °C. Yfh1 and 15 Nlabeled Yfh1 were expressed in E. coli as described by He et al. 14 Since variations of ionic NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript strength lead to significant increases in the melting temperature, we restricted the present investigation to solutions of Yfh1 in salt-free buffers.We recorded 1D and 2D NMR spectra of Yfh1 either in TRIS at pH 7.0 or in HEPES at pH 7.0 in the temperature range −5 to 45 °C. Typically, 0.3-0.5 mM unlabeled or 15 N uniformly labeled protein samples were used. Thanks to t...

Particulate methane monooxygenase (pMMO) is a membrane-bound metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria. The nature of the pMMO active site and the overall metal content are controversial, with spectroscopic and crystallographic data suggesting the presence of a mononuclear copper center, a dinuclear copper center, a trinuclear center, and a diiron center or combinations thereof. Most studies have focused on pMMO from Methylococcus capsulatus (Bath). pMMO from a second organism, Methylosinus trichosporium OB3b, has been purified and characterized by spectroscopic and crystallographic methods. Purified M. trichosporium OB3b pMMO contains ~2 copper ions per 100 kDa protomer. Electron paramagnetic resonance (EPR) spectroscopic parameters indicate that type 2 Cu(II) is present as two distinct species. Extended Xray absorption fine structure (EXAFS) data are best fit with oxygen/nitrogen ligands and reveal a Cu-Cu interaction at 2.52 Å. Correspondingly, X-ray crystallography of M. trichosporium OB3b pMMO shows a dinuclear copper center, similar to that observed previously in the crystal structure of M. capsulatus (Bath) pMMO. There are, however, significant differences between the pMMO structures from the two organisms. A mononuclear copper center present in M. capsulatus (Bath) pMMO is absent in M. trichosporium OB3b pMMO, whereas a metal center occupied by zinc in the M. capsulatus (Bath) pMMO structure is occupied by copper in M. trichosporium OB3b pMMO. These findings extend previous work on pMMO from M. capsulatus (Bath) and provide new insight into the functional importance of the different metal centers.Methanotrophs are eubacteria capable of utilizing methane as their only carbon and energy source. Methanotrophs are divided into several classes on the basis of their cell morphologies, membrane arrangements, and pathways for carbon assimilation. The two most widely studied organisms are the type X methanotroph Methylococcus capsulatus (Bath) and the type II methanotroph Methylosinus trichosporium OB3b (1). The first step of their metabolic pathway is the conversion of methane to methanol by the enzyme methane monooxygenase (MMO), 1 † This work was supported by National Institutes of Health Grants HL13531 (to B.M.H.), DK068139 (to T.L.S.), and GM070473 (to A.C.R.). A.S.H. was the recipient of an NSF graduate research fellowship. ‡ The coordinates of Methylosinus trichosporium OB3b pMMO have been deposited in the Protein Data Bank with accession code 3CHX. *To whom correspondence may be addressed. A.C.R.: tel, 847-467-5301; fax, 847-467-6489; e-mail, amyr@northwestern.edu. T.L.S.: tel, 313-577-5712; fax, 313-577-2765; e-mail, tstemmle@med which exists in both a well-studied, but rarely expressed, soluble iron-containing form (sMMO) (2) and a membrane-bound particulate form (pMMO) (2,3). Although the active site and chemistry of sMMO are well established, the nature of the pMMO catalytic center remains controversial, particularly regarding the number and types of metal i...

Epidermal growth factor receptor (EGFR) signaling is a potent driver of glioblastoma, a malignant and lethal form of brain cancer. Disappointingly, inhibitors targeting receptor tyrosine kinase activity are not clinically effective, and EGFR persists on the plasma membrane to maintain tumor growth and invasiveness. Here we show that endolysosomal pH is critical for receptor sorting and turnover. By functioning as a leak pathway for protons, the Na+/H+ exchanger NHE9 limits luminal acidification to circumvent EGFR turnover and prolong downstream signaling pathways that drive tumor growth and migration. In glioblastoma, NHE9 expression is associated with stem/progenitor characteristics, radiochemoresistance, poor prognosis and invasive growth in vitro and in vivo. Silencing or inhibition of NHE9 in brain tumor initiating cells attenuates tumorsphere formation and improves efficacy of EGFR inhibitor. Thus, NHE9 mediates inside-out control of oncogenic signaling and is a highly druggable target for pan-specific receptor clearance in cancer therapy.