Article ; Online: Differential effects of disulfide bond formation in TEM-1 versus CTX-M-9 β-lactamase.
Protein science : a publication of the Protein Society
2023 Volume 33, Issue 1, Page(s) e4816
Abstract: To investigate how disulfide bonds can impact protein energy landscapes, we surveyed the effects of adding or removing a disulfide in two β-lactamase enzymes, TEM-1 and CTX-M-9. The homologs share a structure and 38% sequence identity, but only TEM-1 ... ...
| Abstract | To investigate how disulfide bonds can impact protein energy landscapes, we surveyed the effects of adding or removing a disulfide in two β-lactamase enzymes, TEM-1 and CTX-M-9. The homologs share a structure and 38% sequence identity, but only TEM-1 contains a native disulfide bond. They also differ in thermodynamic stability and in the number of states populated at equilibrium: CTX-M-9 is two-state whereas TEM-1 has an additional intermediate state. We hypothesized that the disulfide bond is the major underlying determinant for these observed differences in their energy landscapes. To test this, we removed the disulfide bridge from TEM-1 and introduced a disulfide bridge at the same location in CTX-M-9. This modest change to sequence modulates the stabilities-and therefore populations-of TEM-1's equilibrium states and, more surprisingly, creates a novel third state in CTX-M-9. Unlike TEM-1's partially folded intermediate, this third state is a higher-order oligomer with reduced cysteines that retains the native fold and is fully active. Sub-denaturing concentrations of urea shifts the equilibrium to the monomeric form, allowing the disulfide bond to form. Interestingly, comparing the stability of the oxidized monomer with a variant lacking cysteines reveals the disulfide is neither stabilizing nor destabilizing in CTX-M-9, in contrast with the observed stabilization in TEM-1. Thus, we can conclude that engineering disulfide bonds is not always an effective stabilization strategy even when analogous disulfides exist in more stable structural homologs. This study also illustrates how homo-oligomerization can result from a small number of mutations, suggesting complex formation might be easily accessed during a protein family's evolution. |
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| MeSH term(s) | beta-Lactamases/chemistry ; Cysteine ; Disulfides/chemistry ; Escherichia coli Proteins ; Protein Folding | ||||||||||
| Chemical Substances | beta-Lactamases (EC 3.5.2.6) ; CTX-M-9 protein, E coli (EC 3.5.2.-) ; Cysteine (K848JZ4886) ; Disulfides ; Escherichia coli Proteins ; beta-lactamase TEM-1 (EC 3.5.2.6) | ||||||||||
| Language | English | ||||||||||
| Publishing date | 2023-10-14 | ||||||||||
| Publishing country | United States | ||||||||||
| Document type | Journal Article | ||||||||||
| ZDB-ID | 1106283-6 | ||||||||||
| ISSN | 1469-896X ; 0961-8368 | ||||||||||
| ISSN (online) | 1469-896X | ||||||||||
| ISSN | 0961-8368 | ||||||||||
| DOI | 10.1002/pro.4816 | ||||||||||
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| Database | MEDical Literature Analysis and Retrieval System OnLINE |
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