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41.
Unravelling the mechanisms of a protein refolding process based on the association of detergents and co‐solvents 下载免费PDF全文
C. Michaux G. Roussel M. Lopes‐Rodrigues A. Matagne E.A. Perpète 《Journal of peptide science》2016,22(7):485-491
A new technique associating the detergent Sodium Dodecyl Sulphate (SDS) and an alcohol‐type co‐solvent has been set up, showing an unexpected efficiency to refold several types of soluble or membrane proteins. The present contribution deepens the fundamental knowledge on the phenomena underlying this process, considering the refolding of two model peptides featuring the main protein secondary structures: α‐helix and β‐sheet. Their refolding was monitored by fluorescence and circular dichroism, and it turns out that: (i) 100% recovery of the folded structure is observed for both peptides, (ii) the highest the SDS concentration, the more co‐solvent to be added to recover the peptides' native structures, (iii) a high alcohol concentration is required to alter the SDS denaturing properties, (iv) the co‐solvent performance relies on its specific lipophilic–hydrophilic balanced character, (v) the size of the micelle formed by the detergent does not enter the process critical parameters, and (vi) increasing the salt concentration up to 1 M NaCl has a beneficial impact on the process efficiency. These mechanistic aspects will help us to improve the method and extend its application. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
42.
Characterization of a caveolin‐1 mutation associated with both pulmonary arterial hypertension and congenital generalized lipodystrophy 下载免费PDF全文
Bing Han Courtney A. Copeland Yumeko Kawano Erika Berman Rosenzweig Eric D. Austin Layla Shahmirzadi Sha Tang Krishnan Raghunathan Wendy K. Chung Anne K. Kenworthy 《Traffic (Copenhagen, Denmark)》2016,17(12):1297-1312
Congenital generalized lipodystrophy (CGL) and pulmonary arterial hypertension (PAH) have recently been associated with mutations in the caveolin‐1 ( CAV1 ) gene, which encodes the primary structural protein of caveolae. However, little is currently known about how these CAV1 mutations impact caveolae formation or contribute to the development of disease. Here, we identify a heterozygous F160X CAV1 mutation predicted to generate a C‐terminally truncated mutant protein in a patient with both PAH and CGL using whole exome sequencing, and characterize the properties of CAV1 , caveolae‐associated proteins and caveolae in skin fibroblasts isolated from the patient. We show that morphologically defined caveolae are present in patient fibroblasts and that they function in mechanoprotection. However, they exhibited several notable defects, including enhanced accessibility of the C‐terminus of wild‐type CAV1 in caveolae, reduced colocalization of cavin‐1 with CAV1 and decreased stability of both 8S and 70S oligomeric CAV1 complexes that are necessary for caveolae formation. These results were verified independently in reconstituted CAV1 ?/? mouse embryonic fibroblasts. These findings identify defects in caveolae that may serve as contributing factors to the development of PAH and CGL and broaden our knowledge of CAV1 mutations associated with human disease. 相似文献
43.
Christelle Schouppe Jacques Vaillier Renée Venard Michel Rigoulet Jean Velours Francis Haraux 《Journal of bioenergetics and biomembranes》1999,31(2):105-117
The regulation of membrane-bound proton F0F1ATPase by the protonmotive force and nucleotides was studied in yeastmitochondria. Activation occurred in whole mitochondria and the ATPaseactivity was measured just after disrupting the membranes with Triton X-100.Deactivation occurred either in whole mitochondria uncoupled with FCCP, or indisrupted membranes. No effect of Triton X-100 on the ATPase was observed,except a slow reactivation observed only in the absence of MgADP. BothAMPPNP and ATP increased the ATPase deactivation rate, thus indicating thatoccupancy of nucleotidic sites by ATP is more decisive than catalyticturnover for this process. ADP was found to stimulate the energy-dependentATPase activation. ATPase deactivated at the same rate in uncoupled anddisrupted mitochondria. This suggests that deactivation is not controlled byrebinding of some soluble factor, like IF1, but rather by the conversion ofthe F1.IF1 complex into an inactive form. 相似文献
44.
45.
Richard C. Page Sangwon Lee Jacob D. Moore Stanley J. Opella Timothy A. Cross 《Protein science : a publication of the Protein Society》2009,18(1):134-146
The structural characterization of small integral membrane proteins pose a significant challenge for structural biology because of the multitude of molecular interactions between the protein and its heterogeneous environment. Here, the three‐dimensional backbone structure of Rv1761c from Mycobacterium tuberculosis has been characterized using solution NMR spectroscopy and dodecylphosphocholine (DPC) micelles as a membrane mimetic environment. This 127 residue single transmembrane helix protein has a significant (10 kDa) C‐terminal extramembranous domain. Five hundred and ninety distance, backbone dihedral, and orientational restraints were employed resulting in a 1.16 Å rmsd backbone structure with a transmembrane domain defined at 0.40 Å. The structure determination approach utilized residual dipolar coupling orientation data from partially aligned samples, long‐range paramagnetic relaxation enhancement derived distances, and dihedral restraints from chemical shift indices to determine the global fold. This structural model of Rv1761c displays some influences by the membrane mimetic illustrating that the structure of these membrane proteins is dictated by a combination of the amino acid sequence and the protein's environment. These results demonstrate both the efficacy of the structural approach and the necessity to consider the biophysical properties of membrane mimetics when interpreting structural data of integral membrane proteins and, in particular, small integral membrane proteins. 相似文献
46.
Mary Koszelak‐Rosenblum Adam Krol Namrita Mozumdar Kristin Wunsch Adam Ferin Eleanor Cook Christina K. Veatch Raymond Nagel Joseph R. Luft George T. DeTitta Michael G. Malkowski 《Protein science : a publication of the Protein Society》2009,18(9):1828-1839
Elucidating the structures of membrane proteins is essential to our understanding of disease states and a critical component in the rational design of drugs. Structural characterization of a membrane protein begins with its detergent solubilization from the lipid bilayer and its purification within a functionally stable protein‐detergent complex (PDC). Crystallization of the PDC typically occurs by changing the solution environment to decrease solubility and promote interactions between exposed hydrophilic surface residues. As membrane proteins have been observed to form crystals close to the phase separation boundaries of the detergent used to form the PDC, knowledge of these boundaries under different chemical conditions provides a foundation to rationally design crystallization screens. We have carried out dye‐based detergent phase partitioning studies using different combinations of 10 polyethylene glycols (PEG), 11 salts, and 11 detergents to generate a significant amount of chemically diverse phase boundary data. The resulting curves were used to guide the formulation of a 1536‐cocktail crystallization screen for membrane proteins. We are making both the experimentally derived phase boundary data and the 1536 membrane screen available through the high‐throughput crystallization facility located at the Hauptman‐Woodward Institute. The phase boundary data have been packaged into an interactive Excel spreadsheet that allows investigators to formulate grid screens near a given phase boundary for a particular detergent. The 1536 membrane screen has been applied to 12 membrane proteins of unknown structures supplied by the structural genomics and structural biology communities, with crystallization leads for 10/12 samples and verification of one crystal using X‐ray diffraction. 相似文献
47.
Innokentiy Maslennikov Martin Krupa Christopher Dickson Luis Esquivies Katherine Blain Georgia Kefala Senyon Choe Witek Kwiatkowski 《Journal of structural and functional genomics》2009,10(1):25-35
Abstract Bottlenecks in expression, solubilization, purification and crystallization hamper the structural study of integral membrane
proteins (IMPs). Successful crystallization is critically dependent on the purity, stability and oligomeric homogeneity of
an IMP sample. These characteristics are in turn strongly influenced by the type and concentration of the detergents used
in IMP preparation. By utilizing the techniques and analytical tools we earlier developed for the characterization of protein-detergent
complexes (PDCs) [21], we demonstrate that for successful protein extraction from E. coli membrane fractions, the solubilizing detergent associates preferentially to IMPs rather than to membrane lipids. Notably,
this result is contrary to the generally accepted mechanism of detergent-mediated IMP solubilization. We find that for one
particular member of the family of proteins studied (E. coli receptor kinases, which is purified in mixed multimeric states and oligomerizes through its transmembrane region), the protein
oligomeric composition is largely unaffected by a 10-fold increase in protein concentration, by alteration of micelle properties
through addition of other detergents to the PDC sample, or by a 20-fold variation in the detergent concentration used for
solubilization of the IMP from the membrane. We observed that the conditions used for expression of the IMP, which impact
protein density in the membrane, has the greatest influence on the IMP oligomeric structure. Finally, we argue that for concentrating
PDCs smaller than 30 kDa, stirred concentration cells are less prone to over-concentration of detergent and are therefore
more effective than centrifugal ultrafiltration devices. 相似文献
48.
Anne‐Sophie Rivier Guillaume A. Castillon Laetitia Michon Masayoshi Fukasawa Maria Romanova‐Michaelides Nina Jaensch Kentaro Hanada Reika Watanabe 《Traffic (Copenhagen, Denmark)》2010,11(8):1017-1033
Previous studies have shown that yeast glycosylphosphatidylinositol‐anchored proteins (GPI‐APs) and other secretory proteins are preferentially incorporated into distinct coat protein II (COPII) vesicle populations for their transport from the endoplasmic reticulum (ER) to the Golgi apparatus, and that incorporation of yeast GPI‐APs into COPII vesicles requires specific lipid interactions. We compared the ER exit mechanism and segregation of GPI‐APs from other secretory proteins in mammalian and yeast cells. We find that, unlike yeast, ER‐to‐Golgi transport of GPI‐APs in mammalian cells does not depend on sphingolipid synthesis. Whereas ER exit of GPI‐APs is tightly dependent on Sar1 in mammalian cells, it is much less so in yeast. Furthermore, in mammalian cells, GPI‐APs and other secretory proteins are not segregated upon COPII vesicle formation, in contrast to the remarkable segregation seen in yeast. These findings suggest that GPI‐APs use different mechanisms to concentrate in COPII vesicles in the two organisms, and the difference might explain their propensity to segregate from other secretory proteins upon ER exit. 相似文献
49.
Columbus L Lipfert J Klock H Millett I Doniach S Lesley SA 《Protein science : a publication of the Protein Society》2006,15(5):961-975
Structural studies of integral membrane proteins typically rely upon detergent micelles as faithful mimics of the native lipid bilayer. Therefore, membrane protein structure determination would be greatly facilitated by biophysical techniques that are capable of evaluating and assessing the fold and oligomeric state of these proteins solubilized in detergent micelles. In this study, an approach to the characterization of detergent-solubilized integral membrane proteins is presented. Eight Thermotoga maritima membrane proteins were screened for solubility in 11 detergents, and the resulting soluble protein-detergent complexes were characterized with small angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR) spectroscopy, circular dichroism (CD) spectroscopy, and chemical cross-linking to evaluate the homogeneity, oligomeric state, radius of gyration, and overall fold. A new application of SAXS is presented, which does not require density matching, and NMR methods, typically used to evaluate soluble proteins, are successfully applied to detergent-solubilized membrane proteins. Although detergents with longer alkyl chains solubilized the most proteins, further characterization indicates that some of these protein-detergent complexes are not well suited for NMR structure determination due to conformational exchange and protein oligomerization. These results emphasize the need to screen several different detergents and to characterize the protein-detergent complex in order to pursue structural studies. Finally, the physical characterization of the protein-detergent complexes indicates optimal solution conditions for further structural studies for three of the eight overexpressed membrane proteins. 相似文献
50.
Phosphorylation by protein kinase A and dephosphorylation by protein phosphatase 1 modulate the inhibitory activity of phospholamban (PLN), the endogenous regulator of the sarco(endo)plasmic reticulum calcium Ca(2+) ATPase (SERCA). This cyclic mechanism constitutes the driving force for calcium reuptake from the cytoplasm into the myocite lumen, regulating cardiac contractility. PLN undergoes a conformational transition between a relaxed (R) and tense (T) state, an equilibrium perturbed by the addition of SERCA. Here, we show that the single phosphoryl transfer at Ser16 induces a more pronounced conformational switch to the R state in phosphorylated PLN (pPLN). The binding affinity of PLN to SERCA is not affected (K(d) values for the transmembrane domains of pPLN and PLN are approximately 60 microM), supporting the hypothesis that phosphorylation at Ser16 does not dissociate PLN from SERCA. However, the binding surface and dynamics in domain Ib (residues 22-31) change substantially upon phosphorylation. Since PLN can be singly or doubly phosphorylated at Ser16 and Thr17, we propose that these sites remotely control the conformation of domain Ib. These findings constitute a paradigm for how post-translational modifications such as phosphorylation in the cytoplasmic portion of membrane proteins control intramembrane protein-protein interactions. 相似文献