Characterization of a molten globule intermediate during GdnHCl-induced unfolding of RTEM beta-lactamase from Escherichia coli.


Abstract

GdnHCl-induced unfolding and reversible folding of beta-lactamase from E. coli have been investigated by measuring enzymatic activity, fluorescence emission and far-UV circular dichroism as indices of the extent of denaturation. The non-coincidence of far-UV CD and fluorescence data and existence of an inflection point clearly suggest the presence of an equilibrium intermediate. The existence of the equilibrium intermediate at around 1 M is corroborated by its enhanced binding of fluorophobic probe 1,8-ANS. The intermediate was found to have a compact shape as measured by its Stokes radius by size-exclusion chromatography. Furthermore, near-UV CD analysis of this enzymatically inactive intermediate showed a significantly disrupted tertiary structure with only a minor change in the secondary structure, which is a characteristic of typical molten globule states. Estimation of the activation energy from the kinetics of unfolding of the protein monitored by fluorescence and CD suggests that the intermediate may be separated from the native and the unfolded state by a high activation-energy barrier. Study holds ProTherm entries: 10789, 10790 Extra Details: transition 1 beta-lactamase; equilibrium intermediate; molten globule;,protein folding; (Escherichia coli)

Submission Details

ID: gSpLn5WB3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:40 p.m.

Version: 1

Publication Details
Sarkar D;DasGupta C,Biochim. Biophys. Acta (1996) Characterization of a molten globule intermediate during GdnHCl-induced unfolding of RTEM beta-lactamase from Escherichia coli. PMID:8765233
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
2FFY 2006-03-28 1.07 AmpC beta-lactamase N289A mutant in complex with a boronic acid deacylation transition state analog compound SM3
2HDS 2006-11-07 1.16 AmpC beta-lactamase in complex with 4-Methanesulfonylamino benzoic acid
4KZ5 2014-05-21 1.35 Crystal structure of AmpC beta-lactamase in complex with fragment 5 (N-{[3-(2-chlorophenyl)-5-methyl-1,2-oxazol-4-yl]carbonyl}glycine)
4KZB 2014-05-21 1.37 Crystal structure of AmpC beta-lactamase in complex with fragment 50 (N-(methylsulfonyl)-N-phenyl-alanine)
4OKP 2014-05-28 1.37 Crystal structure of AmpC beta-lactamase in complex with the product form of 7-amino-desacetoxycephalosporanic acid
1PI4 2004-02-24 1.39 Structure of N289A mutant of AmpC in complex with SM3, a phenylglyclboronic acid bearing the cephalothin R1 side chain
4LV3 2014-07-30 1.42 AmpC beta-lactamase in complex with (3,5-di-tert-butylphenyl) boronic acid
4KZ4 2014-05-21 1.42 Crystal structure of AmpC beta-lactamase in complex with fragment 60 (2-[(propylsulfonyl)amino]benzoic acid)
4KZ7 2014-05-21 1.43 Crystal structure of AmpC beta-lactamase in complex with fragment 16 ((1R,4S)-4,7,7-trimethyl-3-oxo-2-oxabicyclo[2.2.1]heptane-1-carboxylic acid)
4E3K 2012-09-26 1.43 Crystal structure of AmpC beta-lactamase in complex with a designed 4-tetrazolyl pyridine sulfonamide boronic acid inhibitor
4E3L 2012-09-26 1.43 Crystal structure of AmpC beta-lactamase in complex with a 3-chloro-4-tetrazolyl benzene sulfonamide boronic acid inhibitor
4E3M 2012-09-26 1.44 Crystal structure of AmpC beta-lactamase in complex with a 2-chloro-4-tetrazolyl benzene sulfonamide boronic acid inhibitor
4OLD 2014-05-28 1.48 Crystal structure of AmpC beta-lactamase in complex with the product form of (6R,7R)-7-amino-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid
4E3N 2012-09-26 1.49 Crystal structure of AmpC beta-lactamase in complex with a 2-trifluoromethyl-4-tetrazolyl benzene sulfonamide boronic acid inhibitor
1PI5 2004-02-24 1.49 Structure of N289A mutant of AmpC in complex with SM2, carboxyphenylglycylboronic acid bearing the cephalothin R1 side chain
2HDU 2006-11-07 1.49 AmpC beta-lactamase in complex with 2-acetamidothiophene-3-carboxylic acid
6DPZ 2018-07-04 1.5 X-ray crystal structure of AmpC beta-lactamase with inhibitor
1L0G 2002-08-14 1.5 X-ray Crystal Structure of AmpC S64G Mutant beta-Lactamase
3FKW 2009-03-24 1.5 AmpC K67R mutant apo structure
1KVL 2002-03-13 1.53 X-ray Crystal Structure of AmpC S64G Mutant beta-Lactamase in Complex with Substrate and Product Forms of Cephalothin
1L0D 2002-08-14 1.53 X-ray Crystal Structure of AmpC S64D Mutant beta-Lactamase
3O86 2010-11-03 1.6 Crystal structure of AmpC beta-lactamase in complex with a sulfonamide boronic acid inhibitor
4E3O 2012-09-26 1.6 Crystal structure of AmpC beta-lactamase in complex with a small chloromethyl sulfonamide boronic acid inhibitor
4KZA 2014-05-21 1.6 Crystal structure of AmpC beta-lactamase in complex with fragment 48 (3-(cyclopropylsulfamoyl)thiophene-2-carboxylic acid)
4E3I 2012-09-26 1.6 Crystal structure of AmpC beta-lactamase in complex with a designed 3-carboxyl benzyl sulfonamide boronic acid inhibitor
3IXB 2010-02-16 1.63 X-ray crystal structure of the extended-spectrum AmpC E219K mutant beta-lactamase complexed with benzo(b)thiophene-2-boronic acid (BZB) at 1.63 Angstrom resolution
3O88 2010-11-03 1.64 Crystal structure of AmpC beta-lactamase in complex with a sulfonamide boronic acid inhibitor
3IWI 2010-02-16 1.64 X-ray crystal structure of the extended-spectrum AmpC omega loop insertion (H210AAA) mutant beta-lactamase at 1.64 Angstrom resolution
4JXG 2014-10-29 1.65 Crystal Structure of AmpC beta-lactamase from E. coli in Complex with Oxacillin
4LV0 2014-07-30 1.65 AmpC beta-lactamase in complex with m-aminophenyl boronic acid
4LV2 2014-07-30 1.65 AmpC beta-lactamase in complex with [1-(6-chloropyrimidin-4-yl)-1H-pyrazol-4-yl] boronic acid
1L0F 2002-08-14 1.66 X-ray Crystal Structure of AmpC N152H Mutant beta-Lactamase
4KZ3 2014-05-21 1.67 Crystal structure of AmpC beta-lactamase in complex with fragment 44 (5-chloro-3-sulfamoylthiophene-2-carboxylic acid)
4KZ6 2014-05-21 1.68 Crystal structure of AmpC beta-lactamase in complex with fragment 13 ((2R,6R)-6-methyl-1-(3-sulfanylpropanoyl)piperidine-2-carboxylic acid)
4OLG 2014-05-28 1.71 Crystal structure of AmpC beta-lactamase in complex with covalently bound N-formyl 7-aminocephalosporanic acid
1O07 2003-08-26 1.71 Crystal Structure of the complex between Q120L/Y150E mutant of AmpC and a beta-lactam inhibitor (MXG)
1KE4 2002-07-17 1.72 X-ray crystal structure of AmpC beta-lactamase from E. coli
1MY8 2003-03-04 1.72 AmpC beta-lactamase in complex with an M.carboxyphenylglycylboronic acid bearing the cephalothin R1 side chain
1LLB 2002-10-02 1.72 Crystal Structure Of AmpC beta-Lactamase From E. Coli In Complex With ATMO-penicillin
4KZ9 2014-05-21 1.72 Crystal structure of AmpC beta-lactamase in complex with fragment 41 ((4R,4aS,8aS)-4-phenyldecahydroquinolin-4-ol)
4LV1 2014-07-30 1.74 AmpC beta-lactamase in complex with [1-(3-chlorophenyl)-1H-pyrazol-4-yl] boronic acid
4KG6 2014-10-29 1.75 Crystal Structure of AmpC beta-lactamase N152G Mutant from E. coli
1FSY 2001-03-14 1.75 AMPC BETA-LACTAMASE FROM E. COLI COMPLEXED WITH INHIBITOR CLOXACILLINBORONIC ACID
5JOC 2017-05-03 1.75 Crystal structure of the S61A mutant of AmpC BER
4JXV 2014-04-30 1.76 X-ray crystal structure of AmpC beta-lactamase from E. coli in complex with a non-covalent inhibitor 3-{[2-(4-CARBOXYPHENYL)ETHYL]SULFAMOYL}THIOPHENE-2-CARBOXYLIC ACID (compound 5)
3O87 2010-11-03 1.78 Crystal structure of AmpC beta-lactamase in complex with a sulfonamide boronic acid inhibitor
6DPT 2018-07-04 1.79 X-ray crystal structure of AmpC beta-lactamase with nanomolar inhibitor
3GQZ 2009-04-14 1.8 AmpC beta-lactamase in complex with Fragment-based Inhibitor
3GV9 2009-04-14 1.8 AmpC beta-lactamase in complex with Fragment-based Inhibitor
2P9V 2007-08-14 1.8 Structure of AmpC beta-lactamase with cross-linked active site after exposure to small molecule inhibitor
4E3J 2012-09-26 1.8 Crystal structure of AmpC beta-lactamase in complex with a designed 4-tetrazolyl benzene sulfonamide boronic acid inhibitor
1LL5 2002-11-27 1.8 X-ray crystal structure of AmpC WT beta-lactamase in complex with covalently bound imipenem
3GR2 2009-04-14 1.8 AmpC beta-lactamase in complex with Fragment-based Inhibitor
3GVB 2009-04-14 1.8 AmpC beta-lactamase in complex with Fragment-based Inhibitor
2R9W 2008-04-15 1.8 AmpC beta-lactamase with bound Phthalamide inhibitor
1MXO 2003-03-04 1.83 AmpC beta-lactamase in complex with an m.carboxyphenylglycylboronic acid bearing the cephalothin R1 side chain
1I5Q 2001-06-20 1.83 CRYSTAL STRUCTURE OF THE E. COLI AMPC BETA-LACTAMASE MUTANT N152A COVALENTLY ACYLATED WITH THE INHIBITORY BETA-LACTAM, MOXALACTAM
3IWQ 2010-02-16 1.84 X-ray crystal structure of the extended-spectrum AmpC E219K mutant beta-lactamase at 1.84 Angstrom resolution
3FKV 2009-03-24 1.85 AmpC K67R mutant complexed with benzo(b)thiophene-2-boronic acid (bzb)
2PU2 2008-04-15 1.86 AmpC beta-lactamase with bound Phthalamide inhibitor
1LL9 2002-10-02 1.87 Crystal Structure Of AmpC beta-Lactamase From E. Coli In Complex With Amoxicillin
4KEN 2014-10-29 1.89 Crystal Structure of AmpC beta-lactamase N152G Mutant in Complex with Cefoxitin
4KG2 2014-10-29 1.89 Crystal Structure of AmpC beta-lactamase from E. coli in Complex with Cefotaxime
1FSW 2001-03-14 1.9 AMPC BETA-LACTAMASE FROM E. COLI COMPLEXED WITH INHIBITOR CEPHALOTHINBORONIC ACID
4JXS 2014-05-07 1.9 X-ray crystal structure of AmpC beta-lactamase from E. coli in complex with a non-covalent inhibitor 3-[(4-CARBOXYBENZYL)SULFAMOYL]THIOPHENE-2-CARBOXYLIC ACID (compound 4)
1L0E 2002-08-14 1.9 X-ray Crystal Structure of AmpC K67Q Mutant beta-Lactamase
3GTC 2009-04-14 1.9 AmpC beta-lactamase in complex with Fragment-based Inhibitor
2R9X 2008-04-15 1.9 AmpC beta-lactamase with bound Phthalamide inhibitor
6DPX 2018-07-04 1.9 X-ray crystal structure of AmpC beta-lactamase with inhibitor
3IWO 2010-02-16 1.9 X-ray crystal structure of the extended-spectrum AmpC Y221G mutant beta-lactamase at 1.90 Angstrom resolution
6DPY 2018-07-04 1.91 X-ray crystal structure of AmpC beta-lactamase with inhibitor
1L2S 2002-07-17 1.94 X-ray crystal structure of AmpC beta-lactamase from E. coli in complex with a DOCK-predicted non-covalent inhibitor
1XGI 2005-05-03 1.96 AmpC beta-lactamase in complex with 3-(3-nitro-phenylsulfamoyl)-thiophene-2-carboxylic acid
1XGJ 2005-05-03 1.97 AmpC beta-lactamase in complex with 3-(4-carboxy-2-hydroxy-phenylsulfamoyl)-thiophene-2-carboxylic acid
1IEL 2001-08-15 2.0 Crystal Structure of AmpC beta-lactamase from E. coli in Complex with Ceftazidime
2RCX 2007-11-27 2.0 AmpC Beta-lactamase in complex with (1R)-1-(2-Thiophen-2-yl-acetylamino)-1-(3-(2-carboxyvinyl)-phenyl) methylboronic acid
2PU4 2008-04-15 2.0 AmpC beta-lacamase with bound covalent oxadiazole inhibitor
2BLS 1998-08-12 2.0 AMPC BETA-LACTAMASE FROM ESCHERICHIA COLI
1KVM 2002-03-13 2.06 X-ray Crystal Structure of AmpC WT beta-Lactamase in Complex with Covalently Bound Cephalothin
1GA9 2001-07-25 2.1 CRYSTAL STRUCTURE OF AMPC BETA-LACTAMASE FROM E. COLI COMPLEXED WITH NON-BETA-LACTAMASE INHIBITOR (2, 3-(4-BENZENESULFONYL-THIOPHENE-2-SULFONYLAMINO)-PHENYLBORONIC ACID)
2HDQ 2006-11-07 2.1 AmpC beta-lactamase in complex with 2-carboxythiophene
3BM6 2009-02-17 2.1 AmpC beta-lactamase in complex with a p.carboxyphenylboronic acid
3GSG 2009-04-14 2.1 AmpC beta-lactamase in complex with Fragment-based Inhibitor
4KG5 2014-10-29 2.11 Crystal Structure of AmpC beta-lactamase N152G Mutant in Complex with Cefotaxime
3IXG 2010-02-16 2.14 X-ray crystal structure of the extended-spectrum AmpC T70I mutant beta-lactamase with and without benzo(b)thiophene-2-boronic acid bound at 2.14 Angstrom resolution
1KE3 2002-07-17 2.15 X-ray crystal structure of AmpC beta-lactamase from E. coli in complex with the inhibitor 4,4'-biphenyldiboronic acid
1KDS 2002-07-17 2.15 X-ray crystal structure of AmpC beta-lactamase from E. coli in complex with the inhibitor 3-nitrophenylboronic acid
2I72 2007-09-11 2.2 AmpC beta-lactamase in complex with 5-diformylaminomethyl-benzo[b]thiophen-2-boronic acid
2HDR 2006-11-07 2.2 AmpC beta-lactamase in complex with 4-Amino-3-hydroxybenzoic acid
1FCO 2000-12-04 2.2 CRYSTAL STRUCTURE OF THE E. COLI AMPC BETA-LACTAMASE COVALENTLY ACYLATED WITH THE INHIBITORY BETA-LACTAM, MOXALACTAM
1C3B 1999-11-24 2.25 AMPC BETA-LACTAMASE FROM E. COLI COMPLEXED WITH INHIBITOR, BENZO(B)THIOPHENE-2-BORONIC ACID (BZB)
1KDW 2002-07-17 2.28 X-ray crystal structure of AmpC beta-lactamase from E. coli in complex with the inhibitor 4-carboxyphenylboronic acid
4KZ8 2014-05-21 2.28 Crystal structure of AmpC beta-lactamase in complex with fragment 20 (1,3-diethyl-2-thioxodihydropyrimidine-4,6(1H,5H)-dione)
3IXH 2010-02-16 2.3 X-ray crystal structure of the extended-spectrum AmpC Y221G mutant beta-lactamase in complex with cefotaxime at 2.3 Angstrom resolution
4JXW 2014-05-07 2.3 X-ray crystal structure of AmpC beta-lactamase from E. coli in complex with a non-covalent inhibitor 3-{[3-(4-CARBOXYPHENYL)PROPYL]SULFAMOYL}THIOPHENE-2-CARBOXYLIC ACID (compound 6)
3BLS 1998-08-12 2.3 AMPC BETA-LACTAMASE FROM ESCHERICHIA COLI
1KE0 2002-07-17 2.3 X-ray crystal structure of AmpC beta-lactamase from E. coli in complex with the inhibitor 4-(carboxyvin-2-yl)phenylboronic acid
1IEM 2001-08-15 2.3 Crystal Structure of AmpC beta-lactamase from E. coli in Complex with a Boronic Acid Inhibitor (1, CefB4)
1FCN 2000-12-04 2.35 Crystal Structure of the E. Coli AMPC Beta-Lactamase Mutant Q120L/Y150E Covalently Acylated with the Substrate Beta-Lactam LORACARBEF
1FCM 2000-12-04 2.46 CRYSTAL STRUCTURE OF THE E.COLI AMPC BETA-LACTAMASE MUTANT Q120L/Y150E COVALENTLY ACYLATED WITH THE INHIBITORY BETA-LACTAM, CLOXACILLIN
3GRJ 2009-04-14 2.49 AmpC beta-lactamase in complex with Fragment-based Inhibitor
3IXD 2010-02-16 2.64 X-ray crystal structure of the extended-spectrum AmpC V298E mutant beta-lactamase at 2.64 Angstrom resolution

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Beta-lactamase P00811 AMPC_ECOLI