Application of single-molecule fluorescence resonance energy transfer to maltose transporter

Shiming Huang, Purdue University

Abstract

The maltose transporter, a member of the ATP-binding cassette (ABC) family, translocates maltose and maltodextrins into cell by coupling the energy from ATP hydrolysis. The maltose transporter is composed of a heterodimer of transmembrane subunits MalF and MalG, and a homodimer of nucleotide binding subunits MalK. A periplasmic maltose binding protein delivers maltose or maltodextrins to the translocation pathway and necessary for the ATP hydrolysis. A three-state catalytic model was well established for maltose transporter by the crystal structures in resting state, pre-translocation state and transition state as well as EPR studies. In the studies for this dissertation, single-molecule fluorescence resonance energy transfer (smFRET) based on total internal reflection fluorescence (TIRF) microscopy was applied to study dynamics of transporter immobilized on the surface in real time. The reconstituted Cy3 and Cy5 labeled maltose transporter mutant D128C/D128C in biotinylated nanodisc was tethered on the surface specifically. The interface of the nucleotide-binding domain MalK closed in the presence of vanadate, ATP, maltose-MBP and Mg2+. An E159Q mutant exhibited similar closure pattern in the absence of vanadate. An ATP titration assay was applied to study the dynamics of the transporter. Less proteins closed in the presence of ATP and maltose-MBP and indicated the fluctuations of transporter between resting state, pre-translocation state and transition state. Background fluorescence data was collected on the Cy3 and Cy5 treated cysteine-free maltose transporter. It was not contributed a lot to the real FRET data, but background data was necessary to be concerned during multiple-peak fitting of FRET histogram of D128C/D128C mutant. To express and purify a maltose transporter containing two different versions of MalK, a new plasmid (pET41a) encoding GST affinity tag gene was constructed to express GST-MalK. A TEV protease cut site was inserted between GST tag and MalK to remove the GST containing cysteines. The other version of MalK containing C-terminal polyhistidine tag was constructed to pACYCDUET1 together with MalF and MalG. The maltose transporter containing two different versions of MalK was successfully expressed and purified. By introducing a single-cysteine mutation on the two plasmids containg malK respectively, we obtained a double-cysteine mutant and the two cysteines were at different residues of two MalK chains. To obtain mutants which has maxium distance change between resting state and pre-translocation state, or pre-tranlocation state and transition state, a screening of pairs of residues based on the cystal structures of three conformations and G14C/E108C and V16C/E108C were selected for the smFRET experiment. G14C/E108C showed three FRET states at 0, 0.2, and 0.5 in the absence or prescence of ATP and maltose-MBP. The FRET state peak at 0 decreased and the peak at 0.5 increased in the prescence of ATP and maltose-MBP. The results indicated that the interface of MalKs closed in the transition state. The FRET peak at 0.02 was kept same in both conditions and lower than the other peaks. The result suggested that the pre-translocation state was an intermediate state and short-lived. V16C/E108C only had two peaks in apo or ATP and maltose-MBP bound state, which were located at 0 and 0.48, respectively. The peak at 0 decreased and the other peak at 0.48 increased in the prescence of ATP and maltose-MBP. The results were consistent with G14C/E108C. The failure of V16C/E108C to display a third FRET state was due to the small distance change between resting state and pre-translocation state.

Degree

Ph.D.

Advisors

Hrycyna, Purdue University.

Subject Area

Biochemistry|Physical chemistry

COinS