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Structural and mechanistic insights into ABC-type ECF transporters for vitamin uptake

PhD ceremony:dr. M. (Maria) Dosz-Majsnerowska
When:January 17, 2014
Start:11:00
Supervisor:prof. dr. D.J. (Dirk) Slotboom
Where:Academy building RUG
Faculty:Science and Engineering
Structural and mechanistic insights into ABC-type ECF transporters
for vitamin uptake

The focus of the thesis is on the relationship between structure and mechanism in the ABC-type ECF transporters for vitamin uptake from Lactococcus lactis . Energy Coupling Factor (ECF) transporters are involved in the uptake of vitamins and micronutrients in prokaryotes. They employ integral membrane proteins (S-components) for high affinity substrate binding. S-components form active translocation complexes with the ECF module, which energizes transport of the substrate from ATP hydrolysis. In many cases the ECF module can interact with several different S-components which are unrelated in sequence and bind diverse substrates. This feature enables the transport of chemically different substrates via a common route.
In the first two experimental chapters two S-components specific for biotin (BioY) and thiamin (ThiT) were characterized.
In chapter 2 we presented a high-resolution crystal structure of BioY from L. lactis and performed biochemical analysis of this protein and its homolog from Rhodobacter capsulatus .
In chapter 3 we used EPR spectroscopy, stopped-flow fluorescence spectroscopy and molecular dynamics simulations to determine the structural rearrangements that take place in ThiT from L. lactis upon binding of thiamin.
In the two following experimental chapters the complete ECF transporters were investigated.
In chapter 4 and chapter 5 the mechanism of vitamin transport by ECF transporters from L. lactis was studied in vivo and in vitro (after reconstitution in liposomes), respectively.
This work exemplifies how a combination of structural and biochemical data may bring an important contribution into understanding of biological systems.