Related ArticlesRecombinant locust apolipophorin III: characterization and NMR spectroscopy.
Biochim Biophys Acta. 1998 Jul 31;1393(1):99-107
Authors: Weers PM, Wang J, Van der Horst DJ, Kay CM, Sykes BD, Ryan RO
Apolipophorin III (apoLp-III) from the locust Locusta migratoria is an exchangeable apolipoprotein that reversibly binds to lipoproteins. During lipid binding the protein has been proposed to undergo a major conformational change. To study the mechanism of lipid binding we have cloned and expressed recombinant protein in bacteria, permitting stable isotope enrichment for heteronuclear NMR spectroscopy and site-directed mutagenesis. The cDNA coding for apoLp-III was subcloned into the pET expression vector and transformed into Escherichia coli cells. Induction of expression resulted in the specific appearance of apoLp-III in the cell culture medium, indicating it escaped the bacteria without lysis. The protein was purified from the cell-free supernatant by reversed-phase HPLC, characterized and compared to the natural protein isolated from locust hemolymph. SDS-PAGE revealed the recombinant protein has a molecular mass of approximately 17 kDa, similar to that of deglycosylated natural apoLp-III. Monoclonal antibodies were used to detect recombinant apoLp-III in the cells as well as in cell-free medium of induced bacterial cultures. Amino acid sequencing and analysis confirmed the identity of the recombinant protein as L. migratoria apoLp-III. Circular dichroism spectroscopy of recombinant and natural apoLp-III showed similar spectra, both displaying high contents of alpha-helical secondary structure. Denaturation studies of lipid-free apoLp-III with guanidine hydrochloride showed that both proteins have similar denaturation midpoints and DeltaG values indicating similar protein stability. The natural and recombinant protein were functional in lipoprotein binding assays. Using recombinant protein, uniformly and specifically labeled with 15N-amino acids, two dimensional 1H-15N heteronuclear single quantum correlation spectra were obtained. The spectra revealed excellent chemical shift dispersion in both the 1H and 15N dimensions with a well defined resonance pattern. Studies with 15N-leucine specifically labeled apoLp-III in the presence and absence of the micelle forming lipid, dodecylphosphocholine, provided evidence for a significant conformational change upon lipid association.
In Situ Structural Characterization of a Recombinant Protein in Native Escherichia coli Membranes with Solid-State Magic-Angle-Spinning NMR
In Situ Structural Characterization of a Recombinant Protein in Native Escherichia coli Membranes with Solid-State Magic-Angle-Spinning NMR
Riqiang Fu, Xingsheng Wang, Conggang Li, Adriana N. Santiago-Miranda, Gary J. Pielak and Fang Tian
http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jacsat/0/jacsat.ahead-of-print/ja204062v/aop/images/medium/ja-2011-04062v_0004.gif
Journal of the American Chemical Society
DOI: 10.1021/ja204062v
http://feeds.feedburner.com/~ff/acs/jacsat?d=yIl2AUoC8zA
http://feeds.feedburner.com/~r/acs/jacsat/~4/BuOPwKpaHdw
nmrlearner
Journal club
0
07-27-2011 11:24 AM
Structural characterization of recombinant human myoglobin isoforms by (1)H and (129)Xe NMR and molecular dynamics simulations.
Structural characterization of recombinant human myoglobin isoforms by (1)H and (129)Xe NMR and molecular dynamics simulations.
Structural characterization of recombinant human myoglobin isoforms by (1)H and (129)Xe NMR and molecular dynamics simulations.
Biochim Biophys Acta. 2011 Jul 13;
Authors: Gussoni M, Scorciapino MA, Vezzoli A, Anedda R, Greco F, Ceccarelli M, Casu M
Myoglobin (Mb), the main cytosolic oxygen storage/deliver protein, is also known to interact with different small ligands exerting other fundamental physiological roles. In...
nmrlearner
Journal club
0
07-26-2011 09:30 PM
In Situ Structural Characterization of a Recombinant Protein in Native Escherichia coli Membranes with Solid-State MAS NMR.
In Situ Structural Characterization of a Recombinant Protein in Native Escherichia coli Membranes with Solid-State MAS NMR.
In Situ Structural Characterization of a Recombinant Protein in Native Escherichia coli Membranes with Solid-State MAS NMR.
J Am Chem Soc. 2011 Jul 21;
Authors: Fu R, Wang X, Li C, Santiago-Miranda AN, Pielak GJ, Tian F
The feasibility of using solid state MAS NMR for in situ structural characterization of the LR11 (sorLA) transmembrane domain in native Escherichia coli (E. coli) membranes is presented. LR11 interacts with...
nmrlearner
Journal club
0
07-23-2011 08:54 AM
Expression, purification and NMR characterization of the cyclic recombinant form of the third intracellular loop of the vasopressin type 2 receptor.
Expression, purification and NMR characterization of the cyclic recombinant form of the third intracellular loop of the vasopressin type 2 receptor.
Expression, purification and NMR characterization of the cyclic recombinant form of the third intracellular loop of the vasopressin type 2 receptor.
Protein Expr Purif. 2011 May 13;
Authors: Bellot G, Pascal R, Mendre C, Urbach S, Mouillac B, Déméné H
The vasopressin type 2 (V2R) receptor belongs to the class of G-protein coupled receptors. It is mainly expressed in the membrane of kidney tubules,...
nmrlearner
Journal club
0
05-19-2011 04:20 AM
[NMR paper] Overexpression, purification, and characterization of recombinant Ca-ATPase regulator
Overexpression, purification, and characterization of recombinant Ca-ATPase regulators for high-resolution solution and solid-state NMR studies.
Related Articles Overexpression, purification, and characterization of recombinant Ca-ATPase regulators for high-resolution solution and solid-state NMR studies.
Protein Expr Purif. 2003 Aug;30(2):253-61
Authors: Buck B, Zamoon J, Kirby TL, DeSilva TM, Karim C, Thomas D, Veglia G
Phospholamban (PLB) and Sarcolipin (SLN) are integral membrane proteins that regulate muscle contractility via direct...
nmrlearner
Journal club
0
11-24-2010 09:16 PM
[NMR paper] NMR evidence for a conformational adaptation of apolipophorin III upon lipid associat
NMR evidence for a conformational adaptation of apolipophorin III upon lipid association.
Related Articles NMR evidence for a conformational adaptation of apolipophorin III upon lipid association.
Biochem Cell Biol. 1998;76(2-3):276-83
Authors: Wang J, Sahoo D, Sykes BD, Ryan RO
A characteristic property of amphipathic exchangeable apolipoproteins is an ability to exist alternately in lipid-free and lipid-bound states. In the present study, we have used 1H-15N-heteronuclear single quantum correlation spectroscopy to probe structural changes of...
nmrlearner
Journal club
0
11-17-2010 11:06 PM
[NMR paper] NMR characterization of the full-length recombinant murine prion protein, mPrP(23-231
NMR characterization of the full-length recombinant murine prion protein, mPrP(23-231).
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--linkinghub.elsevier.com-ihub-images-PubMedLink.gif Related Articles NMR characterization of the full-length recombinant murine prion protein, mPrP(23-231).
FEBS Lett. 1997 Aug 18;413(2):282-8
Authors: Riek R, Hornemann S, Wider G, Glockshuber R, Wüthrich K
The recombinant murine prion protein, mPrP(23-231), was expressed in E. coli with uniform 15N-labeling. NMR experiments showed that the previously...
Recombinant locust apolipophorin III: characterization and NMR spectroscopy.
Linear Mode
