One simple step in the identification of the cofactors signals, one giant leap for the solution structure determination of multiheme proteins.
Multiheme proteins play major roles in various biological systems. Structural information on these systems in solution is crucial to understand their functional mechanisms. However, the presence of numerous proton-containing groups in the heme cofactors and the magnetic properties of the heme iron, in particular in the oxidised state, complicates significantly the assignment of the NMR signals. Consequently, the multiheme proteins superfamily is extremely under-represented in structural databases, which constitutes a severe bottleneck in the elucidation of their structural-functional relationships. In this work, we present a strategy that simplifies the assignment of the NMR signals in multiheme proteins and, concomitantly, their solution structure determination, using the triheme cytochrome PpcA from the bacterium Geobacter sulfurreducens as a model. Cost-effective isotopic labeling was used to double label (13C/15N) the protein in its polypeptide chain, with the correct folding and heme post-translational modifications. The combined analysis of 1H-13C HSQC NMR spectra obtained for labeled and unlabeled samples of PpcA allowed a straight discrimination between the heme cofactors and the polypeptide chain signals and their confident assignment. The results presented here will be the foundations to assist solution structure determination of multiheme proteins, which are still very scarce in the literature.
Requimte-CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Campus Caparica, 2829-516 Caparica, Portugal.
Leonor
L
Morgado
Ana P
AP
Fernandes
Yuri Y
YY
Londer
Marta
M
Bruix
Carlos A
CA
Salgueiro
D003067:Coenzymes / Q000737:chemistry*; D003461:Crystallography; D003580:Cytochromes / Q000737:chemistry; D045302:Geobacter / Q000201:enzymology; D006418:Heme / Q000737:chemistry; D006420:Hemeproteins / Q000737:chemistry*; D007553:Isotope Labeling; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*
D003067:Coenzymes; D003580:Cytochromes; D006420:Hemeproteins; D006418:Heme