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.

Summary NHE9 (SLC9A9) is an endosomal cation/proton antiporter with orthologs in yeast and bacteria. Rare, missense substitutions in NHE9 are genetically linked with autism, but have not been functionally evaluated. Here we use evolutionary conservation analysis to build a model-structure of NHE9 based on the crystal structure of bacterial NhaA and use it to screen autism-associated variants in the human population first by phenotype complementation in yeast, followed by functional analysis in primary cortical astrocytes from mouse. NHE9-GFP localizes to recycling endosomes where it significantly alkalinizes luminal pH, elevates uptake of transferrin and the neurotransmitter glutamate, and stabilizes surface expression of transferrin receptor and GLAST transporter. In contrast, autism associated variants L236S, S438P and V176I lack function in astrocytes. Thus, we establish a neurobiological cell model of a candidate gene in autism. Loss of function mutations in NHE9 may contribute to autistic phenotype by modulating synaptic membrane protein expression and neurotransmitter clearance.

The integral membrane enzyme particulate methane monooxygenase (pMMO) converts methane, the most inert hydrocarbon, to methanol under ambient conditions. The 2.8-A resolution pMMO crystal structure revealed three metal sites: a mononuclear copper center, a dinuclear copper center, and a nonphysiological mononuclear zinc center. Although not found in the crystal structure, solution samples of pMMO also contain iron. We have used X-ray absorption spectroscopy to analyze the oxidation states and coordination environments of the pMMO metal centers in as-isolated (pMMO(iso)), chemically reduced (pMMO(red)), and chemically oxidized (pMMO(ox)) samples. X-ray absorption near-edge spectra (XANES) indicate that pMMO(iso) contains both Cu(I) and Cu(II) and that the pMMO Cu centers can undergo redox chemistry. Extended X-ray absorption fine structure (EXAFS) analysis reveals a Cu-Cu interaction in all redox forms of the enzyme. The Cu-Cu distance increases from 2.51 to 2.65 A upon reduction, concomitant with an increase in the average Cu-O/N bond lengths. Appropriate Cu2 model complexes were used to refine and validate the EXAFS fitting protocols for pMMO(iso). Analysis of Fe EXAFS data combined with electron paramagnetic resonance (EPR) spectra indicates that Fe, present as Fe(III), is consistent with heme impurities. These findings are complementary to the crystallographic data and provide new insight into the oxidation states and possible electronic structures of the pMMO Cu ions.

Frataxin, a conserved nuclear encoded mitochondrial protein, plays a direct role in iron-sulfur cluster biosynthesis within the ISC assembly pathway. Humans with frataxin deficiency have Friedreich's ataxia, a neurodegenerative disorder characterized by mitochondrial iron overload and disruption in Fe-S cluster synthesis. Biochemical and genetic studies have shown frataxin interacts with the iron-sulfur cluster assembly scaffold protein (in yeast, there are two: Isu1 and Isu2), indicating frataxin plays a direct role in cluster assembly, possibly by serving as an iron chaperone n the assembly pathway. Here we provide molecular details of how yeast frataxin (Yfh1) interacts with Isu1 as a structural module to better understand the multiprotein complex assembly that completes Fe-S cluster assembly; this complex also includes the cysteine desulfurase (Nfs1 in yeast) and the accessory protein (Isd11), together in the mitochondria. Thermodynamic binding parameters for protein partner and iron binding were measured for the yeast orthologs using isothermal titration calorimetry (ITC). Nuclear magnetic resonance spectroscopy was used to provide the molecular details to understand how Yfh1 interacts with Isu1. X-ray absorption studies were used to electronically and structurally characterize how iron is transferred to Isu1 and then incorporated into a Fe-S cluster. These results were combined with previously published data to generate a structural model for how the Fe-S cluster protein assembly complex can come together to accomplish Fe-S cluster assembly. KeywordsIron Chaperone; Frataxin; Yfh1; Isu1; Nfs1; NMR; ITC and Iron-Sulfur Cluster Assembly Iron-sulfur (Fe-S) clusters are central to life and found in nearly every class of organism (1). These ancient but conserved cofactors are bound to proteins involved in a diverse array of essential functions, ranging from DNA repair to respiration. Since Fe-S cofactors are essential for cell viability, it is no surprise proteins that produce these cofactors are tightly controlled and evolutionarily conserved (2-4). In eukaryotes, the major Fe-S cluster assembly machinery is found in the mitochondria. The process of Fe-S cluster synthesis involves the formation of an Fe-S cluster intermediate on a scaffold protein (ISCU in humans or Isu1 or Isu2 in yeast) and subsequent transfer of the cluster to recipient apo-* To whom correspondence should be sent: Department of Biochemistry and Molecular Biology, Wayne State University, School of Medicine, 540 E. Canfield Ave., Detroit, MI 48201. Telephone: 313-577-5712, Fax: 313-577-2765, tstemmle@med.wayne.edu NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 October 12. proteins. Formation of the Fe-S cluster by Isu1 requires a source of sulfur and a source of iron. The sulfur originates from cysteine via the activity of the cysteine desulfurase Nfs1, which is coordinated with the essential accessory protein Isd11. The source of the iron for Fe-S clusters has remained a mystery. Frataxin (Y...

Autism imposes a major impediment to childhood development and a huge emotional and financial burden on society. In recent years, there has been rapidly accumulating genetic evidence that links the eNHE, a subset of Na+/H+ exchangers that localize to intracellular vesicles, to a variety of neurological conditions including autism, attention deficit hyperactivity disorder (ADHD), intellectual disability, and epilepsy. By providing a leak pathway for protons pumped by the V-ATPase, eNHE determine luminal pH and regulate cation (Na+, K+) content in early and recycling endosomal compartments. Loss-of-function mutations in eNHE cause hyperacidification of endosomal lumen, as a result of imbalance in pump and leak pathways. Two isoforms, NHE6 and NHE9 are highly expressed in brain, including hippocampus and cortex. Here, we summarize evidence for the importance of luminal cation content and pH on processing, delivery and fate of cargo. Drawing upon insights from model organisms and mammalian cells we show how eNHE affect surface expression and function of membrane receptors and neurotransmitter transporters. These studies lead to cellular models of eNHE activity in pre- and post-synaptic neurons and astrocytes, where they could impact synapse development and plasticity. The study of eNHE has provided new insight on the mechanism of autism and other debilitating neurological disorders and opened up new possibilities for therapeutic intervention.

Frataxin, a highly conserved protein found in prokaryotes and eukaryotes, is required for efficient regulation of cellular iron homeostasis. Humans with a frataxin deficiency have the cardio-and neurodegenerative disorder Friedreich's ataxia, commonly resulting from a GAA trinucleotide repeat expansion in the frataxin gene. While frataxin's specific function remains a point of controversy, the general consensus is that the protein assists in controlling cellular iron homeostasis by directly binding iron. This review focuses on the structural and biochemical aspects of iron binding by the frataxin orthologs and outlines molecular attributes that may help explain the protein's role in different cellular pathways.