Competition between DsbA-mediated oxidation and conformational folding of RTEM1 beta-lactamase.


Abstract

Similar to other proteins of the periplasm of Escherichia coli, TEM 1 beta-lactamase contains only a single disulfide bond. It can fold to its native conformation in both the presence and the absence of this disulfide bond. The GdmC1-dependent equilibrium unfolding of beta-lactamase in vitro is well described by a N reversible I reversible U three-state model in which the native protein (N) first reacts to an intermediate of the molten globule type (I) and then to the unfolded state (U). We find that the disulfide bond of beta-lactamase stabilizes I relative to U, but does not change the stability of N relative to I. The I reversible U transition is an extremely rapid reaction for both reduced and oxidized beta-lactamase, but the N reversible I folding kinetics are slow and identical in the presence and the absence of the disulfide bond. This insensitivity of the N reversible I equilibrium and kinetics suggests that the region around the disulfide bond is already native-like folded and is presumably buried in the intermediate I, prior to the slow and rate-limiting events of folding. This was confirmed by measuring the stability of the disulfide bond, which, to a first approximation, is identical in N and I. In native, reduced beta-lactamase, the thiol groups are inaccessible for oxidation by DsbA, but at the stage of the molten globule intermediate I oxidation is still possible, because I is in fast exchange with the unfolded protein U. The introduction of the disulfide bond into beta-lactamase by DsbA competes with conformational folding at the stage of the final slow steps in the folding of the reduced protein. The major problem in the oxidation of proteins with one or two disulfide bonds (such as beta-lactamase) is not the formation of incorrect disulfide bonds, but the premature burial of the thiol groups by the rapid conformational folding of the reduced protein. DsbA, the major thiol/ disulfide isomerase of the bacterial periplasm, meets this problem. It is a very strong oxidant, and its reaction with cysteine residues in unfolded proteins is extremely fast. Study holds ProTherm entries: 4842, 4843, 4844, 4845 Extra Details: additive : EDTA(2 mM),N -> I disulfide bond; native conformation; molten globule;,thiol groups

Submission Details

ID: SHqF9tzQ3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:27 p.m.

Version: 1

Publication Details
Frech C;Wunderlich M;Glockshuber R;Schmid FX,Biochemistry (1996) Competition between DsbA-mediated oxidation and conformational folding of RTEM1 beta-lactamase. PMID:8784194
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