Electrospray‐ionization mass spectrometry of intact intrinsic membrane proteins

Whitelegge, Julian P.; Gundersen, Cameron B.; Faull, Kym F.

doi:10.1002/pro.5560070619

Cited by 192 publications

(192 citation statements)

References 51 publications

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“…Column eluent was directed to an ion-spray source of a triple-quadrupole mass spectrometer (API IIIϩ; PE Sciex/Applied Biosystems, Foster City, CA) tuned and calibrated as described in ref. 35, yielding mass accuracy of 0.01%.…”

Section: Methodsmentioning

confidence: 99%

Metal-free superoxide dismutase forms soluble oligomers under physiological conditions: A possible general mechanism for familial ALS

Banci

Bertini

Durazo³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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225

262

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Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder selectively affecting motor neurons; 90% of the total cases are sporadic, but 2% are associated with mutations in the gene coding for the antioxidant enzyme copper-zinc superoxide dismutase (SOD1). The causes of motor neuron death in ALS are poorly understood in general, but for SOD1-linked familial ALS, aberrant oligomerization of SOD1 mutant proteins has been strongly implicated. In this work, we show that wild-type human SOD1, when lacking both its metal ions, forms large, stable, soluble protein oligomers with an average molecular mass of Ϸ650 kDa under physiological conditions, i.e., 37°C, pH 7.0, and 100 M protein concentration. It further is shown here that intermolecular disulfide bonds are formed during oligomerization and that Cys-6 and Cys-111 are implicated in this bonding. The formation of the soluble oligomers was monitored by their ability to enhance the fluorescence of thioflavin T, a benzothiazole dye that increases in fluorescence intensity upon binding to amyloid fibers, and by disruption of this binding upon addition of the chaotropic agent guanidine hydrochloride. Our results suggest a general, unifying picture of SOD1 aggregation that could operate when wild-type or mutant SOD1 proteins lack their metal ions. Although we cannot exclude other mechanisms in SOD1-linked familial ALS, the one proposed here has the strength of explaining how a large and diverse set of SOD1 mutant proteins all could lead to disease through the same mechanism.amyloid ͉ neurodegeneration ͉ protein aggregation ͉ amyotrophic lateral sclerosis ͉ protein misfolding P rotein oligomerization, aggregation, and formation of insoluble amyloid deposits commonly are observed in neurodegenerative diseases, but the factors initiating and modulating the abnormal protein-protein interactions that lead to oligomerization remain elusive (1, 2). Metal ions frequently have been implicated in these phenomena, but how exactly they are involved remains unclear (3). Over 114 different variants of human copper-zinc superoxide dismutase (Cu 2 Zn 2 SOD1) have been linked to the neurodegenerative disease familial ALS (FALS) by a gain-of-function mechanism (4-6). Although the exact cellular sites and mechanisms of toxicity are unknown, aberrant SOD1 protein oligomerization has been strongly implicated in disease causation (7,8). Several recent publications have presented compelling evidence that abnormal disulfide cross-linking of ALS-mutant SOD1 plays a role in this oligomerization, and disulfide-linked SOD1 multimers have been detected in neural tissues of SOD1-ALS transgenic mice that are presumed to be components of higher-molecular-weight species or intermediates in their formation (7, 9-11).Wild-type (WT) human SOD1 is an exceptionally stable protein in its holo form and, although some of the ALS-mutant SOD1 proteins are severely destabilized by their mutations, others largely retain the stability of WT SOD1 (4). In the fully demetallated (apo) states, some ...

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Section: Methodsmentioning

confidence: 99%

Metal-free superoxide dismutase forms soluble oligomers under physiological conditions: A possible general mechanism for familial ALS

Banci

Bertini

Durazo³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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225

262

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“…Site-directed mutations were introduced to recombinant WT bacteriorhodopsin and their identity confirmed by mass spectrometry (47). Equilibrium unfolding curves, kinetic data, and chevron plots were collected.…”

Section: Methodsmentioning

confidence: 99%

Stable folding core in the folding transition state of an α-helical integral membrane protein

Curnow

Bartolo²,

Moreton³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Defining the structural features of a transition state is important in understanding a folding reaction. Here, we use Φ-value and double mutant analyses to probe the folding transition state of the membrane protein bacteriorhodopsin. We focus on the final C-terminal helix, helix G, of this seven transmembrane helical protein. Φ-values could be derived for 12 amino acid residues in helix G, most of which have low or intermediate values, suggesting that native structure is disrupted at these amino acid positions in the transition state. Notably, a cluster of residues between E204 and M209 all have Φ-values close to zero. Disruption of helix G is further confirmed by a low Φ-value of 0.2 between residues T170 on helix F and S226 on helix G, suggesting the absence of a native hydrogen bond between helices F and G. Φ-values for paired mutations involved in four interhelical hydrogen bonds revealed that all but one of these bonds is absent in the transition state. The unstructured helix G contrasts with Φ-values along helix B that are generally high, implying native structure in helix B in the transition state. Thus helix B seems to constitute part of a stable folding nucleus while the consolidation of helix G is a relatively late folding event. Polarization of secondary structure correlates with sequence position, with a structured helix B near the N terminus contrasting with an unstructured C-terminal helix G.

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“…MS has been used previously to analyze purified PSII complexes (Whitelegge et al, 1998;Zheleva et al, 1998), but no systematic analysis of chloroplast proteins has been conducted. In this study, we present detailed reproducible two-dimensional electrophoresis maps of the lumenal and peripheral proteins of the thylakoids from pea.…”

Section: Introductionmentioning

confidence: 99%

Proteomics of the Chloroplast: Systematic Identification and Targeting Analysis of Lumenal and Peripheral Thylakoid Proteins

Peltier

Friso

Kalume³

et al. 2000

Plant Cell

336

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The soluble and peripheral proteins in the thylakoids of pea were systematically analyzed by using two-dimensional electrophoresis, mass spectrometry, and N-terminal Edman sequencing, followed by database searching. After correcting to eliminate possible isoforms and post-translational modifications, we estimated that there are at least 200 to 230 different lumenal and peripheral proteins. Sixty-one proteins were identified; for 33 of these proteins, a clear function or functional domain could be identified, whereas for 10 proteins, no function could be assigned. For 18 proteins, no expressed sequence tag or full-length gene could be identified in the databases, despite experimental determination of a significant amount of amino acid sequence. Nine previously unidentified proteins with lumenal transit peptides are presented along with their full-length genes; seven of these proteins possess the twin arginine motif that is characteristic for substrates of the TAT pathway. Logoplots were used to provide a detailed analysis of the lumenal targeting signals, and all nuclear-encoded proteins identified on the two-dimensional gels were used to test predictions for chloroplast localization and transit peptides made by the software programs ChloroP, PSORT, and SignalP. A combination of these three programs was found to provide a useful tool for evaluating chloroplast localization and transit peptides and also could reveal possible alternative processing sites and dual targeting. The potential of proteomics for plant biology and homology-based searching with mass spectrometry data is discussed.

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Electrospray‐ionization mass spectrometry of intact intrinsic membrane proteins

Cited by 192 publications

References 51 publications

Metal-free superoxide dismutase forms soluble oligomers under physiological conditions: A possible general mechanism for familial ALS

Metal-free superoxide dismutase forms soluble oligomers under physiological conditions: A possible general mechanism for familial ALS

Stable folding core in the folding transition state of an α-helical integral membrane protein

Proteomics of the Chloroplast: Systematic Identification and Targeting Analysis of Lumenal and Peripheral Thylakoid Proteins

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