Transmembrane segment enhanced labeling as a tool for the backbone assignment of α-helical membrane proteins

Reckel, Sina; Sobhanifar, Solmaz; Schneider, Birgit; Junge, Friederike; Schwarz, Dániel; Durst, Florian; Löhr, Frank; Güntert, Peter; Bernhard, Frank; Dötsch, Volker

doi:10.1073/pnas.0710843105

Cited by 35 publications

(16 citation statements)

References 28 publications

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“…S1). To obtain backbone assignments, we used a combination of standard triple resonance experiments with uniform as well as TMS labeled samples (30), resulting in the assignment of 84% of the total resonances and 90% of resonances (E356-I467) excluding the N terminus which contains an unstructured loop. The combined chemical shift and NOE information revealed the presence of six α-helical regions (Fig.…”

Section: Resultsmentioning

confidence: 99%

Structural investigation of the C-terminal catalytic fragment of presenilin 1

Sobhanifar

Schneider

Löhr

et al. 2010

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

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The γ-secretase complex has a decisive role in the development of Alzheimer's disease, in that it cleaves a precursor to create the amyloid β peptide whose aggregates form the senile plaques encountered in the brains of patients. Γ-secretase is a member of the intramembrane-cleaving proteases which process their transmembrane substrates within the bilayer. Many of the mutations encountered in early onset familial Alzheimer's disease are linked to presenilin 1, the catalytic component of γ-secretase, whose active form requires its endoproteolytic cleavage into N-terminal and C-terminal fragments. Although there is general agreement regarding the topology of the N-terminal fragment, studies of the C-terminal fragment have yielded ambiguous and contradictory results that may be difficult to reconcile in the absence of structural information. Here we present the first structure of the C-terminal fragment of human presenilin 1, as obtained from NMR studies in SDS micelles. The structure reveals a topology where the membrane is likely traversed three times in accordance with the more generally accepted nine transmembrane domain model of presenilin 1, but contains unique structural features adapted to accommodate the unusual intramembrane catalysis. These include a putative half-membrane-spanning helix N-terminally harboring the catalytic aspartate, a severely kinked helical structure toward the C terminus as well as a soluble helix in the assumed-to-be unstructured N-terminal loop.cell-free protein expression | gamma secretase | intramembrane proteolysis | membrane protein structure A lzheimer's disease is the most common form of dementia and affects more than 25 million people worldwide. The most characteristic histological feature of Alzheimer's disease is the presence of long, insoluble amyloid fibrils composed of amyloid β (Aβ) peptide which, either alone or as reservoirs for soluble Aβ oligomers (1, 2), appear to be the primary species responsible for the massive neuronal injury presented in patients. Aβ generation is categorized under an unusual physiological phenomenon termed regulated intramembrane proteolysis. Here, the amyloid precursor protein first sheds its ectodomain mediated by β-secretase. The remaining membrane-bound C-terminal fragment is subsequently processed at a γ-cleavage site by the γ-secretase complex, a multisubunit protease whose minimal essential components include presenilin 1 (PS1) or presenilin-2 (PS2), anterior pharynx-defective, nicastrin, and presenilin enhancer 2 (3). The pathological relevance of this final step lies in the observation that γ-cleavage is variable and can occur after three distinct positions, 38, 40, and 42, whose selection influences the self-aggregating potential of the secreted Aβ peptide. Aβ42, although the minor species, appears to show the strongest potency for oligomerization and represents the majority of Aβ in amyloid plaques (4). Over 150 familial Alzheimer's disease associated mutations (www.molgen.ua.ac.be/ADMutations) have been linked to PS1, the catalyt...

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Section: Resultsmentioning

confidence: 99%

Structural investigation of the C-terminal catalytic fragment of presenilin 1

Sobhanifar

Schneider

Löhr

et al. 2010

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

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“…Recent development of efficient molecular biological tools for the expression of integral membrane proteins [7][8][9][10][11] and improvement of their stability 12,13 have opened new routes towards high-resolution structural studies by X-ray crystallography and NMR spectroscopy. Besides providing information on molecular structures, NMR offers unique possibilities to examine membrane protein dynamics on a large range of timescales.…”

Section: Introductionmentioning

confidence: 99%

Solution State NMR Structure and Dynamics of KpOmpA, a 210 Residue Transmembrane Domain Possessing a High Potential for Immunological Applications

Renault

Saurel

Czaplicki

et al. 2009

Journal of Molecular Biology

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“…Deuteration 13 and transverse relaxation optimized spectroscopy (TROSY) pulse sequences 14 circumvent relaxation effects that hinder NMR spectral quality of larger biomolecules (>25 kDa). In addition, assignment of resonances in regions with significant spectral overlap can be addressed with specific amino acid labeling [15][16][17] as well as compensating for the loss of proton-proton distance restraints due to deuteration. 18,19 Other methods, such as residual dipolar couplings 20 and paramagnetic relaxation enhancement, 21 have provided additional structural restraints crucial for large a-helical proteins (e.g., a-helical membrane proteins in detergent micelles).…”

Section: Introductionmentioning

confidence: 99%

“…18,19 Other methods, such as residual dipolar couplings 20 and paramagnetic relaxation enhancement, 21 have provided additional structural restraints crucial for large a-helical proteins (e.g., a-helical membrane proteins in detergent micelles). With these advances, six solution NMR b-barrel membrane protein structures (varying in the number of b-strands (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) and molecular weight (16-31 kDa) have been determined. [1][2][3][4][5][6] In this study, the difficulties of assigning Opa 60 , a b-barrel membrane protein from Neisseria gonorrhoeae, 22 are outlined and strategies for circumventing these challenges are demonstrated.…”

Section: Introductionmentioning

confidence: 99%

Solution NMR resonance assignment strategies for β‐barrel membrane proteins

Fox

Columbus

2013

Protein Science

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Membrane proteins in detergent micelles are large and dynamic complexes that present challenges for solution NMR investigations such as spectral overlap and line broadening. In this study, multiple methods are introduced to facilitate resonance assignment of b-barrel membrane proteins using Opa 60 from Neisseria gonorrhoeae as a model system. Opa 60 is an eight-stranded b-barrel with long extracellular loops (~63% of the protein) that engage host receptors and induce engulfment of the bacterium. The NMR spectra of Opa 60 in detergent micelles exhibits significant spectral overlap and resonances corresponding to the loop regions had variable line widths, which interfered with a complete assignment of the protein. To assign the b-barrel residues, trypsin cleavage was used to remove much of the extracellular loops while preserving the detergent solubilized b-barrel. The removal of the loop resonances significantly improved the assignment of the Opa 60 b-barrel region (97% of the resonances corresponding to the b-barrel and periplasmic turns were assigned). For the loop resonance assignments, two strategies were implemented; modulating temperature and synthetic peptides. Lowering the temperature broadened many peaks beyond detection and simplified the spectra to only the most dynamic regions of the loops facilitating 27 loop resonances to be assigned. To further assign functionally important and unstructured regions of the extracellular loops, a synthetic 20 amino acid peptide was synthesized and had nearly complete spectral overlap with the full-length protein allowing 17 loop resonances to be assigned. Collectively, these strategies are effective tools that may accelerate solution NMR structure determination of b-barrel membrane proteins.

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Transmembrane segment enhanced labeling as a tool for the backbone assignment of α-helical membrane proteins

Abstract: cell-free expression ͉ NMR ͉ stable isotope labeling ͉ protein structure analysis ͉ peak overlap

Cited by 35 publications

References 28 publications

Structural investigation of the C-terminal catalytic fragment of presenilin 1

Structural investigation of the C-terminal catalytic fragment of presenilin 1

Solution State NMR Structure and Dynamics of KpOmpA, a 210 Residue Transmembrane Domain Possessing a High Potential for Immunological Applications

Solution NMR resonance assignment strategies for β‐barrel membrane proteins

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