Isolation, folding and structural investigations of the amino acid transporter OEP16

Ni, Da Qun; Zook, James; Klewer, Douglas A.; Nieman, Ronald A.; Soll, Juergen; Fromme, Petra

doi:10.1016/j.pep.2011.08.004

Cited by 9 publications

(17 citation statements)

References 61 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…High yields of recombinantly expressed 15 N-enriched OEP16 in minimal media have been previously reported [6]. These results give a starting point for expression and purification of the uniformly 15 N, 13 C, 2 H-labeled protein needed for 3D NMR experiments.…”

Section: Introductionmentioning

confidence: 55%

“…For the NMR experiments, U- 13 C, 15 N-labeled and 80% perdeuterated recombinant OEP16 was expressed by using a slightly modified procedure compared to Ni et al [6]. A 5-mL preculture was prepared in Lysogeny Broth (LB) media that was allowed to incubate at 310 K overnight, shaking at 200 rpm.…”

Section: Methodsmentioning

confidence: 99%

“…Protein expression was induced at OD 600 = 0.8 using 1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) [6], and allowing it to incubate for an additional 5 h. Purification and reconstitution of OEP16 in SDS micelles was carried out using previously developed methods [6]. Protein was prepared for the NMR experiments by diluting the concentrated samples in a 10% 2 H 2 O buffer (100 mM NaCl, 20 mM NaH 2 PO 4 pH 6.5, 1 mM β-mercaptoethanol (BME), 1 mM ethylenediaminetetraacetic acid (EDTA), 10% (v/v) glycerol, 0.4% (w/v) SDS, and 0.02% (w/v) NaN 3 ).…”

Section: Methodsmentioning

confidence: 99%

“…One integral membrane protein that has shown promise as a target for NMR study is the outer envelope protein with a molecular mass of 16 kDa (OEP16) from the chloroplast membrane [6]. OEP16 is a transmembrane (TM) protein that shares some sequence homologies (52%) to a putative protein from the mitochondrial membrane translocase of the inner membrane (TIM) that may be part of the protein translocase complex (UniProtKB Accession number: ABF95523.1).…”

Section: Introductionmentioning

confidence: 99%

“…A later model suggested a four TM-helix bundle [9], which contrasts with the textbook view that nearly all outer membrane channels form β-barrels. Yet improved CD spectral data support the four-helix bundle hypothesis [6]. Dimers have been considered from cross-linking studies [7]; electron micrographs have led to the suggestion of trimer formation [10]; and moreover hexameric and higher oligomeric forms have been hypothesized from gel filtration data [6].…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

High-Resolution NMR Reveals Secondary Structure and Folding of Amino Acid Transporter from Outer Chloroplast Membrane

et al. 2013

Self Cite

View full text Add to dashboard Cite

Solving high-resolution structures for membrane proteins continues to be a daunting challenge in the structural biology community. In this study we report our high-resolution NMR results for a transmembrane protein, outer envelope protein of molar mass 16 kDa (OEP16), an amino acid transporter from the outer membrane of chloroplasts. Three-dimensional, high-resolution NMR experiments on the 13C, 15N, 2H-triply-labeled protein were used to assign protein backbone resonances and to obtain secondary structure information. The results yield over 95% assignment of N, HN, CO, Cα, and Cβ chemical shifts, which is essential for obtaining a high resolution structure from NMR data. Chemical shift analysis from the assignment data reveals experimental evidence for the first time on the location of the secondary structure elements on a per residue basis. In addition T 1Z and T2 relaxation experiments were performed in order to better understand the protein dynamics. Arginine titration experiments yield an insight into the amino acid residues responsible for protein transporter function. The results provide the necessary basis for high-resolution structural determination of this important plant membrane protein.

show abstract

Section: Introductionmentioning

confidence: 55%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

High-Resolution NMR Reveals Secondary Structure and Folding of Amino Acid Transporter from Outer Chloroplast Membrane

et al. 2013

Self Cite

View full text Add to dashboard Cite

show abstract

Refolding of urea‐denatured α‐chymotrypsin by protein‐folding liquid chromatography

Sun

Zhang

et al. 2012

Biomedical Chromatography

View full text Add to dashboard Cite

An approach for re-folding denatured proteins during proteome research by protein folding liquid chromatography (PFLC) is presented. Standard protein, α-chymotrypsin (α-Chy), was selected as a model protein and hydrophobic interaction chromatography was performed as a typical PFLC; the three different α-Chy states - urea-denatured (U state), its folded intermediates (M state) and nature state (N state) - were studied during protein folding. Based on the test by matrix-assisted laser desorption/ionization time of flight mass spectrometry and bioactivity, only one stable M state of the α-Chy was identified and then it was prepared for further investigation. The specific bioactivity of the refolded α-Chy was found to be higher than that of commercial α-Chy as the urea concentration in the sample solution ranged from 1.0 to 3.0 m; the highest specific bioactivity at urea concentration was 1.0 m, indicating the possibility for re-folding some proteins that have partially or completely lost their bioactivity, as a dilute urea solution was employed for dissolving the sample. The experiment showed that the peak height of its M state increased with increasing urea concentration, and correspondingly decreased in the amount of the refolded α-Chy. When the urea concentration reached 6.0 m, the unfolded α-Chy could not be refolded at all.

show abstract

Solution Properties of Alkyl β‐D‐Maltosides

Zhencao

Chen

et al. 2019

J Surfact & Detergents

View full text Add to dashboard Cite

Alkyl β‐D‐maltosides are an important class of sugar‐based nonionic surfactants and have been widely studied. Nevertheless, it is still necessary to investigate further their amphiphilic structure‐surface property relationships. In this article, we reported a series of properties of synthetic alkyl β‐D‐maltosides (6a–6i, n = 6–18) including their hydrophilic–lipophilic balance (HLB) number, water solubility, hygroscopicity, moisture‐retention capacity, foaming ability, surface tension, thermotropic phase behavior, and skin irritation. Their HLB number and water solubility decreased with increasing alkyl chain length. Hexyl β‐D‐maltoside exhibited the strongest hygroscopicity and moisture‐retention capacity. Decyl β‐D‐maltoside and dodecyl β‐D‐maltoside possessed excellent foaming power and foaming stability. Furthermore, the critical micelle concentration (CMC) of alkyl β‐D‐maltoside (6a–6g, n = 6–14) and their surface tension at CMC decreased with increasing alkyl chain length. At last, alkyl β‐D‐maltosides (6a–6g) should be considered as safe surfactants by the skin irritation assessment.

show abstract

Isolation, folding and structural investigations of the amino acid transporter OEP16

Cited by 9 publications

References 61 publications

High-Resolution NMR Reveals Secondary Structure and Folding of Amino Acid Transporter from Outer Chloroplast Membrane

High-Resolution NMR Reveals Secondary Structure and Folding of Amino Acid Transporter from Outer Chloroplast Membrane

Refolding of urea‐denatured α‐chymotrypsin by protein‐folding liquid chromatography

Solution Properties of Alkyl β‐D‐Maltosides

Contact Info

Product

Resources

About