Abstract：
:We have developed a simple and efficient method for creation of novel protein functions in an existing protein scaffold. The in vitro coevolution method involves design of a hypothetical pathway for the target function followed by stepwise directed evolution of the corresponding protein along the pathway. As a test case, this strategy was used to engineer variants of human estrogen receptor alpha ligand-binding domain (hERalphaLBD) with novel corticosterone activity. Two steroids, testosterone and progesterone, that provide a progressive structural bridge between 17beta-estradiol and corticosterone, were chosen to assist the directed evolution of hERalphaLBD. A total of approximately 10(6) variants were screened in four rounds of random mutagenesis, resulting in two hERalphaLBD variants that respond to corticosterone. Creation of this new ligand activity required the presence of four simultaneous mutations. In addition, several required mutations were located outside the ligand binding pocket and yet exerted important action on ligand binding. Our results demonstrate the ability of in vitro coevolution to create novel protein function that is difficult or impossible to achieve by existing protein engineering approaches and also shed light on the natural evolution of nuclear hormone receptors. This in vitro coevolution approach should provide a powerful, broadly applicable tool for engineering biological molecules and systems with novel functions.
journal_name
J Mol Bioljournal_title
Journal of molecular biologyauthors
Chen Z,Zhao Hdoi
10.1016/j.jmb.2005.02.070keywords:
subject
Has Abstractpub_date
2005-05-20 00:00:00pages
1273-82issue
5eissn
0022-2836issn
1089-8638pii
S0022-2836(05)00268-8journal_volume
348pub_type
杂志文章abstract：:Alpha-actinin is the major F-actin crosslinking protein in both muscle and non-muscle cells. We report the crystal structure of the actin binding domain of human muscle alpha-actinin-3, which is formed by two consecutive calponin homology domains arranged in a "closed" conformation. Structural studies and available bi...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/j.jmb.2005.01.002
更新日期：2005-04-22 00:00:00
abstract：:YibK is a 160 residue homodimeric protein belonging to the SPOUT class of methyltransferases. Proteins in this group all display a unique topological feature; the backbone polypeptide chain folds to form a deep trefoil knot. Such knotted structures were completely unpredicted, it being thought impossible for a protein...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/j.jmb.2004.12.055
更新日期：2005-03-11 00:00:00
abstract：:Prions are composed largely, if not entirely, of the scrapie isoform of the prion protein (PrPSc). Conversion of the cellular isoform (PrPC) to PrPSc is accompanied by a diminution in the alpha-helical content and an increase in the beta-sheet structure. To investigate the structural basis of this transition, peptide ...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1006/jmbi.1995.0395
更新日期：1995-07-21 00:00:00
abstract：:Glycosaminoglycans (GAGs) are related to multiple biological functions and diseases. There is growing evidence that GAG concentration and sulfate content increase with age. The destabilizing mutation A546T in the corneal protein TGFBIp leads to lattice-type corneal dystrophy, but symptoms only appear in the fourth dec...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/j.jmb.2017.07.014
更新日期：2017-09-01 00:00:00
abstract：:GTP and Ca2+, two well-known modulators of intracellular signaling pathways, control a structural/functional switch between two vital and mutually exclusive activities, cross-linking and Galpha activity, in the same enzyme. The enzyme, a brain-derived tissue-type transglutaminase (TGase), was recently cloned by us in ...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1006/jmbi.1998.2052
更新日期：1998-10-02 00:00:00
abstract：:Crystals of the mouse major urinary protein (MUP) and rat alpha-2u globulin (AMG) have been grown from solutions of polyethylene glycol 3350 and CdCl2, respectively. The crystals differ both in their morphologies and space groups but have very similar unit cell sizes. AMG crystallized in P2(1) (a = 56.6 A, b = 103.8 A...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/0022-2836(91)90258-8
更新日期：1991-04-20 00:00:00
abstract：:In bacteriophages of the cystovirus family, the polymerase complex (PX) encodes a 75-kDa RNA-directed RNA polymerase (P2) that transcribes the double-stranded RNA genome. Also a constituent of the PX is the essential protein P7 that, in addition to accelerating PX assembly and facilitating genome packaging, plays a re...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/j.jmb.2014.04.028
更新日期：2014-07-15 00:00:00
abstract：:Bacteriorhodopsin is the smallest autonomous light-driven proton pump. Proposals as to how it achieves the directionality of its trans-membrane proton transport fall into two categories: accessibility-switch models in which proton transfer pathways in different parts of the molecule are opened and closed during the ph...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/s0022-2836(03)00903-3
更新日期：2003-10-03 00:00:00
abstract：:The preparation of hybrid histone octamers with wheat histone H2A variants replacing chicken H2A in the chicken octamer is described. The fidelity of the reconstituted hybrid octamers was confirmed by dimethyl suberimidate cross-linking. Polyglutamic-acid-mediated assembly of these octamers on long DNA and subsequent ...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/0022-2836(91)90268-b
更新日期：1991-04-20 00:00:00
abstract：:The structural fluctuations specific to self-assembled biological systems have been investigated further with ultrasonic techniques by using two strains of tobacco mosaic virus (TMV), as well as the helical aggregate of the common strain protein and subassemblies of it. We confirmed our earlier conclusion that protein...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/0022-2836(85)90328-6
更新日期：1985-01-05 00:00:00
abstract：:Human pancreatic alpha-amylase has been isolated using a glycogen affinity precipitation procedure and crystallized in a form suitable for high resolution three-dimensional X-ray crystallographic analyses. Crystals are of the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions of a = 53.04 A, b = 74.80 A...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1006/jmbi.1993.1221
更新日期：1993-04-05 00:00:00
abstract：:The thermosome from Thermoplasma acidophilum is a type II chaperonin composed of eight alpha and eight beta subunits. The genes encoding the two types of subunit were co-expressed in Escherichia coli and the alpha8/beta8 complex purified from the cell extract. The isolated complex showed steady-state ATPase properties...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/j.jmb.2005.01.066
更新日期：2005-04-22 00:00:00
abstract：:Crystals of a relatively unspecific NAD(P)H dehydrogenase from human erythrocytes suitable for X-ray analysis have been grown. They belong to space group P4(1)2(1)2 or its enantiomorph P4(3)2(1)2 with unit cell dimensions: a = b = 79.3 A and c = 38.1 A. The asymmetric unit contains one molecule of Mr 18,000. ...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/0022-2836(84)90093-7
更新日期：1984-04-25 00:00:00
abstract：:The co-crystal structure of the MS2 coat protein dimer with its RNA operator reveals eight amino acid side-chains contacting seven of the RNA phosphates. These eight amino acids and five nearby control positions were individually changed to an alanine residue and the binding affinities of the mutant proteins to the RN...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/j.jmb.2005.11.046
更新日期：2006-02-24 00:00:00
abstract：:The ATPase activity of the DNA packaging protein gp16 (gene product 16) of bacteriophage phi 29 was studied in the completely defined in-vitro assembly system. ATP was hydrolyzed to ADP and Pi in the packaging reaction that included purified proheads, DNA-gp3 and gp16. Approximately one molecule of ATP was used in the...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/0022-2836(87)90121-5
更新日期：1987-09-20 00:00:00
abstract：:The pre-steady-state kinetics of GTP hydrolysis catalysed by elongation factor G and ribosomes from Escherichia coli has been investigated by the method of quenched-flow. The GTPase activities either uncoupled from or coupled to the ribosomal translocation process were characterized under various experimental conditio...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/0022-2836(86)90495-x
更新日期：1986-06-20 00:00:00
abstract：:alpha 1-Antitrypsin is the archetypal member of the serine proteinase inhibitor or serpin superfamily. Members of the family show structural homology based on a dominant A beta-sheet and a mobile reactive centre loop. Our recent crystal structure of alpha 1-antitrypsin stabilized with a point mutation showed the loop ...
journal_title：Journal of molecular biology
pub_type： 杂志文章,评审
doi：10.1006/jmbi.1997.1458
更新日期：1998-01-23 00:00:00
abstract：:Specific cysteine residues at possible methyl acceptor sites of the Ada protein of Escherichia coli were converted to other amino acids by site-directed mutagenesis of the cloned ada gene of E. coli. Ada protein with the cysteine residue at 321 replaced by alanine was capable of accepting the methyl group from the met...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/0022-2836(88)90137-4
更新日期：1988-05-20 00:00:00
abstract：:The nucleus is sculpted toward various morphologies during cellular differentiation and development. Alterations in nuclear shape often result in changes to chromatin organization and genome function. This is thought to be reflective of its role as a cellular mechanotransducer. Recent evidence has highlighted the impo...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/j.jmb.2014.09.008
更新日期：2015-02-13 00:00:00
abstract：:The crystal structure of glycosyltrehalose trehalohydrolase from the hyperthermophilic archaeum Sulfolobus solfataricus KM1 has been solved by multiple isomorphous replacement. The enzyme is an alpha-amylase (family 13) with unique exo-amylolytic activity for glycosyltrehalosides. It cleaves the alpha-1,4 glycosidic b...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1006/jmbi.2000.3977
更新日期：2000-08-11 00:00:00
abstract：:Prions are the agents of a series of lethal neurodegenerative diseases. They are composed largely, if not entirely, of the host-encoded prion protein (PrP), which can exist in the cellular isoform PrP(C) and the pathological isoform PrP(Sc). The conformational change of the alpha-helical PrP(C) into beta-sheet-rich Pr...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/j.jmb.2007.11.035
更新日期：2008-02-15 00:00:00
abstract：:FtsH (HflB) is an ATP-dependent protease found in prokaryotic cells, mitochondria and chloroplasts. Here, we have identified, in the carboxy-terminal region of FtsH (HfIB), a short alpha helix predicted of forming a coiled-coil, leucine zipper, structure. This region appears to be structurally conserved. The presence ...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1006/jmbi.2000.3767
更新日期：2000-06-16 00:00:00
abstract：:We investigate the suggestion that aromatic rings can act as hydrogen-bond acceptors in proteins, by an analysis of 55 non-homologous high-resolution protein chain structures. Approximately 10% of interactions between sp2 hybridized nitrogen atoms, from either side-chains or main-chains, and phenylalanine or tyrosine ...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1006/jmbi.1994.1370
更新日期：1994-06-03 00:00:00
abstract：:In-vivo studies have demonstrated that adenovirus type 2 and adenovirus type 4 have different DNA sequence requirements for the initiation of DNA replication. To investigate the basis of these differences an in-vitro system has been developed which will faithfully initiate adenovirus type 4 DNA replication. A plasmid ...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/0022-2836(88)90438-x
更新日期：1988-05-05 00:00:00
abstract：:The DnaB protein is the primary replicative helicase in Escherichia coli, and the active form of the protein is a hexamer. It has been reported that the protein forms a ring with strong 3-fold symmetry, which was suggested to be a trimer of dimers. We show that under different conditions, using either ATP, ATP gamma S...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1006/jmbi.1996.0297
更新日期：1996-05-31 00:00:00
abstract：:The dominant assumption central to most treatments for sickle cell anemia has been that replacement of sickle hemoglobin (HbS) by fetal hemoglobin (HbF) would have major clinical benefit. Using laser photolysis, we have measured polymerization kinetics including rates of homogeneous and heterogeneous nucleation on mix...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/j.jmb.2005.02.006
更新日期：2005-04-15 00:00:00
abstract：:As regulators of ubiquitous biological processes, serine proteases can cause disease states when inappropriately expressed or regulated, and are thus rational targets for inhibition by drugs. Recently we described a new inhibition mechanism applicable for the development of potent, selective small molecule serine prot...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1006/jmbi.1999.3071
更新日期：1999-09-24 00:00:00
abstract：:The motion of the atoms in the small protein bovine pancreatic trypsin inhibitor has been simulated for about 60 picoseconds using two different potential energy functions. In one known as HHL the hydrogen bond is purely electrostatic, in the other, known as L79, the hydrogen bond is a directional O . . . H interactio...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/s0022-2836(83)80304-0
更新日期：1983-08-15 00:00:00
abstract：:Although chemical signaling during embryogenesis is readily addressed by a plethora of available techniques, the developmental functions of ionic signaling are still poorly understood. It is increasingly realized that bioelectric events in nonneural cells are critical for pattern regulation, but their study has been h...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/j.jmb.2019.10.029
更新日期：2020-01-17 00:00:00
abstract：:To define the structural basis for cofactor binding to membrane proteins, we introduce a manageable model system, which allows us, for the first time, to study the influence of individual transmembrane helices and of single amino acid residues on the assembly of a transmembrane cytochrome. In vivo as well as in vitro ...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/j.jmb.2005.05.016
更新日期：2005-07-22 00:00:00
