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
1C3B 1999-07-27T00:00:00+0000 2.25 AMPC BETA-LACTAMASE FROM E. COLI COMPLEXED WITH INHIBITOR, BENZO(B)THIOPHENE-2-BORONIC ACID (BZB)
1FCM 2000-07-18T00:00:00+0000 2.46 CRYSTAL STRUCTURE OF THE E.COLI AMPC BETA-LACTAMASE MUTANT Q120L/Y150E COVALENTLY ACYLATED WITH THE INHIBITORY BETA-LACTAM, CLOXACILLIN
1FCN 2000-07-18T00:00:00+0000 2.35 Crystal Structure of the E. Coli AMPC Beta-Lactamase Mutant Q120L/Y150E Covalently Acylated with the Substrate Beta-Lactam LORACARBEF
1FCO 2000-07-19T00:00:00+0000 2.2 CRYSTAL STRUCTURE OF THE E. COLI AMPC BETA-LACTAMASE COVALENTLY ACYLATED WITH THE INHIBITORY BETA-LACTAM, MOXALACTAM
1FSW 2000-09-11T00:00:00+0000 1.9 AMPC BETA-LACTAMASE FROM E. COLI COMPLEXED WITH INHIBITOR CEPHALOTHINBORONIC ACID
1FSY 2000-09-11T00:00:00+0000 1.75 AMPC BETA-LACTAMASE FROM E. COLI COMPLEXED WITH INHIBITOR CLOXACILLINBORONIC ACID
1GA9 2000-11-29T00:00:00+0000 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)
1I5Q 2001-02-28T00:00:00+0000 1.83 CRYSTAL STRUCTURE OF THE E. COLI AMPC BETA-LACTAMASE MUTANT N152A COVALENTLY ACYLATED WITH THE INHIBITORY BETA-LACTAM, MOXALACTAM
1IEL 2001-04-10T00:00:00+0000 2.0 Crystal Structure of AmpC beta-lactamase from E. coli in Complex with Ceftazidime
1IEM 2001-04-10T00:00:00+0000 2.3 Crystal Structure of AmpC beta-lactamase from E. coli in Complex with a Boronic Acid Inhibitor (1, CefB4)

Relevant UniProtKB Entries

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