Abstract:
:We recently developed a display method for the directed evolution of integral membrane proteins in the inner membrane of Escherichia coli for higher expression and stability. For the neurotensin receptor 1, a G-protein-coupled receptor (GPCR), we had evolved a mutant with a 10-fold increase in functional expression that largely retains wild-type binding and signaling properties and shows higher stability in detergent-solubilized form. We have now evolved three additional human GPCRs. Unmodified wild-type receptor cDNA was subjected to successive cycles of mutagenesis and fluorescence-activated cell sorting, and functional expression could be increased for all three GPCR targets. We also present a new stability screening method in a 96-well assay format to quickly identify evolved receptors showing increased thermal stability in detergent-solubilized form and rapidly evaluate them quantitatively. Combining the two methods turned out to be very powerful; even for the most challenging GPCR target--the tachykinin receptor NK(1), which is hardly expressed in E. coli and cannot be functionally solubilized--receptor mutants that are functionally expressed at 1 mg/l levels in E. coli and are stable in detergent solution could be quickly evolved. The improvements result from cumulative small changes in the receptor sequence. This combinatorial approach does not require preconceived notions for designing mutations. Our results suggest that this method is generally applicable to GPCRs. Existing roadblocks in structural and biophysical studies can now be removed by providing sufficient quantities of correctly folded and stable receptor protein.
journal_name
J Mol Bioljournal_title
Journal of molecular biologyauthors
Dodevski I,Plückthun Adoi
10.1016/j.jmb.2011.02.051subject
Has Abstractpub_date
2011-05-13 00:00:00pages
599-615issue
4eissn
0022-2836issn
1089-8638pii
S0022-2836(11)00230-0journal_volume
408pub_type
杂志文章abstract::Native state 1H NMR resonance assignments for 125 of the 129 residues of equine lysozyme have enabled measurement of the hydrogen exchange kinetics for over 60 backbone amide and three tryptophan indole hydrogen atoms in the native state. Native holo equine lysozyme hydrogen exchange protection factors are as large as...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1006/jmbi.1997.0996
更新日期:1997-05-23 00:00:00
abstract::Protein domains represent the basic evolutionary units that form proteins. Domain duplication and shuffling by recombination are probably the most important forces driving protein evolution and hence the complexity of the proteome. While the duplication of whole genes as well as domain-encoding exons increases the abu...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2004.11.050
更新日期:2005-02-11 00:00:00
abstract::The binding of tryptophan repressor (TrpR) to its operators was examined quantitatively using in vitro and in vivo methods. DNA sequence requirements for 1:1 and tandem 2:1 (TrpR:DNA) binding in various sequence contexts were studied. The results indicate that the optimal half-site sequence for recognition by one heli...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1006/jmbi.1996.0232
更新日期:1996-04-26 00:00:00
abstract::Co-operativity in ATP hydrolysis by GroEL can be described by a model in which each ring of GroEL is in equilibrium between a low (T) and high (R) affinity state for ATP. According to this model, the GroEL double-ring is in equilibrium between three states: TT, TR and RR. In order to find out which states bind non-fol...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1006/jmbi.1996.0028
更新日期:1996-01-26 00:00:00
abstract::The papillomavirus E2 transcription and replication factors bind to the DNA consensus ACCGN(4)CGGT sequence (E2-BS), through both direct and indirect readout mechanisms. The two symmetric half-sites ACCG.CGGT are highly conserved in the genomes and are hydrogen bound with E2. Although E2 does not contact the N4 spacer...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2004.03.078
更新日期:2004-06-11 00:00:00
abstract::Quantitative measurements of local chromatin accessibility to DNase I in 15-day chicken embryo erythrocyte nuclei have been performed using a range of nuclease concentrations and real-time TaqMan PCR to monitor the loss of short ( approximately 80 bp) amplicons. At the beta-globin locus, well-established DNase I hyper...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2008.12.048
更新日期:2009-02-20 00:00:00
abstract::The nuclear receptor pregnane X receptor (PXR) acts as a sensor for a broad variety of natural and synthetic lipophilic compounds, such as bile acids and rifampicin, and regulates the expression of proteins that are involved in the metabolism and transport of these compounds. PXR binds as a heterodimer with the retino...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2004.12.003
更新日期:2005-02-18 00:00:00
abstract::Switching between the active (ATP and DNA bound) and inactive conformations of the homologous recombination RecA protein is regulated by ATP hydrolysis. First, we use the homologous pairing domain of RecA derived from its mobile loop L2 to show that the interaction of this random coil peptide with the gamma-phosphate ...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1006/jmbi.2000.4163
更新日期:2000-11-10 00:00:00
abstract::UMP kinase (UMPK), the enzyme responsible for microbial UMP phosphorylation, plays a key role in pyrimidine nucleotide biosynthesis, regulating this process via feed-back control and via gene repression of carbamoyl phosphate synthetase (the first enzyme of the pyrimidine biosynthesis pathway). We present crystal stru...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2005.07.045
更新日期:2005-09-16 00:00:00
abstract::Fumarase C purified from Escherichia coli has been crystallized in the presence of polyethylene glycol in both a citrate buffer at pH 5.3 and a 3-(4-morpholino)-propanesulfonic acid buffer at pH 7.5 yielding two crystal forms. An orthorhombic C222(1) form was obtained in citrate at pH 5.3 and an orthorhombic I222 form...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1006/jmbi.1993.1264
更新日期:1993-05-05 00:00:00
abstract::Oligonucleotides can bind to double-stranded DNA in a sequence-specific manner to form triple helices. Uniformly modified, pyrimidine-rich oligodeoxyribonuclotides containing internucleosidic N3'-P5' phosphoramidate linkages are known to form very stable triplexes with their DNA target. Psoralen-conjugated triple heli...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1006/jmbi.2000.4386
更新日期:2001-02-09 00:00:00
abstract::C-tailed membrane proteins insert into the bilayer post-translationally because the hydrophobic anchor segment leaves the ribosome at the end of translation. Nevertheless, we find here evidence that the targeting of SciP to the membrane of Escherichia coli occurs co-translationally since signal elements in the N-termi...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2016.09.001
更新日期:2016-10-09 00:00:00
abstract::Transcription regulation relies in the molecular interplay between the RNA polymerase (RNAP) and regulatory factors. Phage phi29 promoters A2c, A2b and A3 are coordinately regulated by the transcriptional regulator protein p4 and the histone-like protein p6. This study shows that protein p4 binds simultaneously to fou...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2003.12.039
更新日期:2004-02-13 00:00:00
abstract::We report a new vector, pEVOL, for the incorporation of unnatural amino acids into proteins in Escherichia coli using evolved Methanocaldococcus jannaschii aminoacyl-tRNA synthetase(s) (aaRS)/suppressor tRNA pairs. This new system affords higher yields of mutant proteins through the use of both constitutive and induci...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2009.10.030
更新日期:2010-01-15 00:00:00
abstract::Replacement of residues 228-230 or 228-232 of subdomain 4 in Dictyostelium actin with the corresponding Tetrahymena sequence (QTA to KAY replacement: half chimera-1; QTAAS to KAYKE replacement: full chimera) leads to a higher Ca(2+)-activation of the regulated acto-myosin subfragment-1 ATPase activity. The ratio of AT...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1006/jmbi.1999.3467
更新日期:2000-02-18 00:00:00
abstract::Bleomycin (Bm)-binding protein, designated BLMA, which is a Bm resistance determinant from Bm-producing Streptomyces verticillus, was crystallized in a form suitable for X-ray diffraction analysis. The diffraction intensity data were collected up to a resolution of 1.5 A with a merging R-value of 0.054 at a completene...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1006/jmbi.1999.3404
更新日期:2000-01-28 00:00:00
abstract::Eicosanoids are potent lipid mediators involved in central physiological processes such as hemostasis, renal function and parturition. When formed in excess, eicosanoids become critical players in a range of pathological conditions, in particular pain, fever, arthritis, asthma, cardiovascular disease and cancer. Eicos...
journal_title:Journal of molecular biology
pub_type: 杂志文章,评审
doi:10.1016/j.jmb.2020.07.020
更新日期:2020-08-21 00:00:00
abstract::Deoxyribonucleoside kinases (dNKs) catalyze the transfer of a phosphoryl group from ATP to a deoxyribonucleoside (dN), a key step in DNA precursor synthesis. Recently structural information concerning dNKs has been obtained, but no structure of a bacterial dCK/dGK enzyme is known. Here we report the structure of such ...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2006.12.010
更新日期:2007-03-09 00:00:00
abstract::Bulges are common structural motifs in RNA secondary structure and are thought to play important roles in RNA-protein and RNA-drug interactions. Adenosine bases are the most commonly occurring unpaired base in double helical RNA secondary structures. The solution conformation and dynamics of a 25-nucleotide RNA duplex...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1006/jmbi.2000.3931
更新日期:2000-07-28 00:00:00
abstract::Streptomyces griseus protease B (SGPB) has primary specificity for large hydrophobic residues. The protease is secreted in a promature form, and autocatalytic removal of the propeptide is essential for activity. We genetically substituted the P1 Leu at the promature junction of SGPB with Phe, Met, or Val and monitored...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1006/jmbi.1996.0159
更新日期:1996-03-29 00:00:00
abstract::Membrane-embedded pyrophosphatase (M-PPase) hydrolyzes pyrophosphate to drive ion (H+ and/or Na+) translocation. We determined crystal structures and functions of Vigna radiata M-PPase (VrH+-PPase), the VrH+-PPase-2Pi complex and mutants at hydrophobic gate (residue L555) and exit channel (residues T228 and E225). Ion...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2019.03.009
更新日期:2019-04-05 00:00:00
abstract::We report here the structure of BstYI, an "intermediate" type II restriction endonuclease with overlapping sequence specificities to BamHI and BglII. BstYI, a thermophilic endonuclease, recognizes and cleaves the degenerate hexanucleotide sequence 5'-RGATCY-3' (where R=A or G and Y=C or T), cleaving DNA after the 5'-R...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2004.02.074
更新日期:2004-05-07 00:00:00
abstract::Rigid-body docking has become quite successful in predicting the correct conformations of binary protein complexes, at least when the constituent proteins do not undergo large conformational changes upon binding. However, determining whether two given proteins interact is a more difficult problem. Successful docking p...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2008.08.002
更新日期:2008-10-24 00:00:00
abstract::The MotA protein of Escherichia coli is a component of the flagellum that functions, together with MotB, in transmembrane proton conduction. MotA and MotB are believed to form the stator of the flagellar motor. They are integral membrane proteins; MotA has a large (ca 22 kDa) domain in the cytoplasm, and MotB a much s...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1006/jmbi.1997.1316
更新日期:1997-10-24 00:00:00
abstract::The high resolution structures of bovine pancreatic trypsin inhibitor refined in two distinct crystal forms have been compared. One of the structures was a result of new least-squares X-ray refinement of data from crystal form I, while the other was the joint X-ray/neutron structure of crystal form II. After superposi...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/0022-2836(87)90633-4
更新日期:1987-01-05 00:00:00
abstract::The secondary structure of ribonuclease T1 (RNase T1) in aqueous solution and its temperature-induced structural changes have been investigated by Fourier-transform infrared (FT-IR) spectroscopy. 13 to 14% alpha-helix and 32 to 33% beta-sheet were estimated from the resolution-enhanced FT-IR spectra, in agreement with...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1006/jmbi.1993.1442
更新日期:1993-08-05 00:00:00
abstract::The MCM motor of the CMG helicase powers ahead of the eukaryotic replication machinery to unwind DNA, in a process that requires ATP hydrolysis. The reconstitution of DNA replication in vitro has established the succession of events that lead to replication origin activation by the MCM and recent studies have started ...
journal_title:Journal of molecular biology
pub_type: 杂志文章,评审
doi:10.1016/j.jmb.2016.01.024
更新日期:2016-05-08 00:00:00
abstract::The nucleotide sequence was obtained for the hypE gene in the cluster of structural and accessory genes required for the assembly and functioning of the membrane-bound, dimeric, (NiFe)hydrogenase in Azotobacter vinelandii. The hypE gene encodes a polypeptide of 341 amino acid residues which is rich in alanine, glycine...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1006/jmbi.1994.1149
更新日期:1994-02-11 00:00:00
abstract::The result of binding the effectors ATP and CTP to aspartate transcarbamylase was studied by X-ray solution scattering. Binding of substrate analogues produces a substantial change in the solution scattering curve, allowing us to monitor the proportion of the different quaternary structure states present in solution. ...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/0022-2836(85)90190-1
更新日期:1985-09-05 00:00:00
abstract::The structure of three consecutive laminin-type EGF-like (LE) modules of mouse laminin gammma1 chain, gamma1III3-5 (positions 738 to 899), has been determined by multiple isomorphous replacement in a crystal of space group p6(4)22 (a=b=74.57 angstroms, c = 185.11 angstroms and gamma = 120 degrees). The crystal structu...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1006/jmbi.1996.0191
更新日期:1996-04-05 00:00:00