Format

Send to

Choose Destination
Protein Sci. 2019 Nov;28(11):2004-2010. doi: 10.1002/pro.3728. Epub 2019 Oct 14.

A simple protocol to characterize bacterial cell-envelope lipoproteins in a native-like environment.

Author information

1
Laboratorio de Metaloproteínas, Instituto de Biología Molecular y Celular de Rosario (IBR, CONICET-UNR), Rosario, Argentina.
2
Área Biofísica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina.

Abstract

Physiological conditions in living cells are strictly regulated to allow, optimize, and coordinate biological processes. The bacterial cell envelope is the compartment where the communication with the external environment takes place. This involves membrane proteins, key players in many biological processes that ensure bacterial survival. The biochemical characterization of membrane proteins, either integral, lipidated or peripheral is challenging due to their mixed protein-lipid nature, making it difficult to purify and obtain considerable amounts of samples. In contrast to integral membrane proteins, lipidated proteins are usually purified as truncated soluble versions, neglecting the impact of the membrane environment. Here we report a simple and robust protocol to characterize bacterial lipidated proteins in spheroplasts from Escherichia coli using a β-lactamase as a model. The Metallo-β-lactamase NDM-1 is an enzyme anchored to the inner leaflet of the outer membrane of Gram-negative bacteria. Kinetic parameters and stability of the lipidated NDM-1 and the soluble unbound version (NDM-1 C26A) were measured in spheroplasts and periplasm, respectively. These studies revealed that membrane anchoring increases the KM of the enzyme, consequently decreasing the catalytic efficiency, while not affecting its kinetic stability. This approach can be used to characterize lipidated proteins avoiding the purification step while mimicking its native environment. This approach also helps in filling the gap between in vitro and in vivo studies.

KEYWORDS:

bacterial outer membrane; kinetic stability; lipidated proteins; lipidic environment; spheroplasts

PMID:
31518027
PMCID:
PMC6798131
[Available on 2020-11-01]
DOI:
10.1002/pro.3728

Supplemental Content

Full text links

Icon for Wiley
Loading ...
Support Center