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Biol Chem ; 404(7): 727-737, 2023 06 27.
Artículo en Inglés | MEDLINE | ID: mdl-37185095

RESUMEN

The essential Escherichia coli ATPase MsbA is a lipid flippase that serves as a prototype for multi drug resistant ABC transporters. Its physiological function is the transport of lipopolisaccharides to build up the outer membranes of Gram-negative bacteria. Although several structural and biochemical studies of MsbA have been conducted previously, a detailed picture of the dynamic processes that link ATP hydrolysis to allocrit transport remains elusive. We report here for the first time time-resolved Fourier transform infrared (FTIR) spectroscopic measurements of the ATP binding and ATP hydrolysis reaction of full-length MsbA and determined reaction rates at 288 K of k 1 = 0.49 ± 0.28 s-1 and k 2 = 0.014 ± 0.003 s-1, respectively. We further verified these rates with photocaged NPEcgAppNHp where only nucleotide binding was observable and the negative mutant MsbA-H537A that showed slow hydrolysis (k 2 < 2 × 10-4 s-1). Besides single turnover kinetics, FTIR measurements also deliver IR signatures of all educts, products and the protein. ADP remains protein-bound after ATP hydrolysis. In addition, the spectral changes observed for the two variants MsbA-S378A and MsbA-S482A correlated with the loss of hydrogen bonding to the γ-phosphate of ATP. This study paves the way for FTIR-spectroscopic investigations of allocrite transport in full-length MsbA.


Asunto(s)
Proteínas Bacterianas , Proteínas de Escherichia coli , Proteínas Bacterianas/metabolismo , Espectroscopía Infrarroja por Transformada de Fourier , Hidrólisis , Adenosina Trifosfato/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo
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