An evaluation of detergents for NMR structural studies of membrane proteins

Krueger-Koplin, Ray D.; Sorgen, Paul L.; Krueger-Koplin, Suzanne T.; Rivera-Torres, Iván O.; Cahill, Sean M.; Hicks, David; Grinius, Leo; Krulwich, Terry A.; Girvin, Mark E.

doi:10.1023/b:jnmr.0000012875.80898.8f

Cited by 172 publications

(167 citation statements)

References 38 publications

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“…kinase (35)] long-range NOE restraints. To obtain long-range distance restraints, we employed paramagnetic relaxation enhancement (PRE) (36)(37)(38)(39). Monocysteine mutants were obtained by mutating the two native cysteines of CTF to alanines [shown to have no effect on PS1 activity (40,41)] and introducing a single cysteine at 13 different positions residing in both loops and helices ( 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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“…With the increasing level of activity in the field of solution NMR of membrane proteins, many new suggestions for lipids and combinations of lipids are emerging [52,53]. Since the key solution NMR measurements are performed with samples that are weakly aligned in polyacryl-amide gels, it is also necessary to simultaneously optimize the conditions for the various gel samples.…”

Section: Detergent Selection-mentioning

confidence: 99%

NMR of membrane proteins in micelles and bilayers: The FXYD family proteins

Franzin

Gong

Thai

et al. 2007

Methods

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Determining the atomic resolution structures of membrane proteins is of particular interest in contemporary structural biology. Helical membrane proteins constitute one-third of the expressed proteins encoded in a genome, many drugs have membrane-bound proteins as their receptors, and mutations in membrane proteins result in human diseases. Although integral membrane proteins provide daunting technical challenges for all methods of protein structure determination, nuclear magnetic resonance (NMR) spectroscopy can be an extremely versatile and powerful method for determining their structures and characterizing their dynamics, in lipid environments that closely mimic the cell membranes. Once milligram amounts of isotopically labeled protein are expressed and purified, micelle samples can be prepared for solution NMR analysis, and lipid bilayer samples can be prepared for solid-state NMR analysis. The two approaches are complementary and can provide detailed structural and dynamic information. This paper describes the steps for membrane protein structure determination using solution and solid-state NMR. The methods for protein expression and purification, sample preparation and NMR experiments are described and illustrated with examples from the FXYD proteins, a family of regulatory subunits of the Na, KATPase.

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“…These well established standard procedures have been used, for example, to prepare the E.coli transporter EmrE for solution state NMR (Schwaiger et al 1998). Unfortunately, α-helical membrane proteins often have a low spectral dispersion (Krueger-Koplin et al 2004), and therefore uniformly labelled proteins yield very crowded NMR spectra with many overlapping peaks. To circumvent this problem, selective labelling of single amino acid types can be achieved with a defined medium (synthetic rich) containing all amino acids (Muchmore et al 1989).…”

Section: Sample Preparationmentioning

confidence: 99%

“…Long term stability is also a prerequisite for solution state NMR, where the best detergent is usually judged by the quality of the protein NMR spectrum. Using this strategy, a thorough screen of 25 different detergents was performed on a Staphylococcus aureus Smr (Krueger- Koplin et al 2004) where a number of promising candidates were found, such as, for example, 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-RAC-(1-glycerol)] (LPPG). Despite the fact that the protein detergent complexes appeared to be larger than 100 kDa, the rotational correlation time corresponded to that of a 15-20 kDa protein tumbling isotropically in solution.…”

Section: Solubilisation Purification Choice Of Detergentsmentioning

confidence: 99%

Investigating transport proteins by solid state NMR

Basting

Lehner

Lorch

et al. 2006

Naunyn Schmied Arch Pharmacol

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Transporters form an interesting and complex class of membrane proteins. Many of them are potential drug targets due to their role in translocation of ions, small molecules and peptides across the membrane or due to their role in multidrug resistance. Hence elucidating their structure and mechanism is of great importance and may lead to a host of new drugs and methods to alter or inhibit their function. Solid state NMR is an emerging technique for investigating transport proteins. Along with other biochemical and biophysical techniques solid state NMR can provide data on drug binding, protein dynamics and structure at the interface between structural biology and functional analysis. Here, we review solid state NMR applications to primary active and secondary transporters involved in translocation of small molecules. We discuss current experimental limitations and give an overall perspective on how the technique may be used to address some pertinent questions relevant to transporters.

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An evaluation of detergents for NMR structural studies of membrane proteins

Cited by 172 publications

References 38 publications

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

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

NMR of membrane proteins in micelles and bilayers: The FXYD family proteins

Investigating transport proteins by solid state NMR

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