Figure 4

FRET based time course measurement to study effect of AuNP conjugation on Gα _i1 basal nucleotide uptake and hydrolysis. Fluorescently-labeled nucleotide (mant-GTP) was used to do FRET studies for monitoring rates of nucleotide exchange. (A) Displays effect of non-covalently conjugated AuNP- Gα_i1 on FRET based measurements. The solid line represents 0 nM AuNP; Short-short-short line represents 0.1 nM AuNP; Dash-dot-dash line represents 0.2 nM AuNP; Dotted line represents 0.3 nM AuNP; Long-short line represents 0.4 nM AuNP. (B) Displays effect of N-terminal covalent conjugation on FRET based measurements. The solid line represents covalently conjugated AuNP-Gα_i1. In all the experiments, a final concentration of 200 nM Gα_i1 was taken in a quartz cuvette containing 5 mM Hepes-Na (pH 8.0), 10 mM NaCl, 0.5 mM MgCl₂, and 1 μM GDP. Time course FRET was monitored by exciting the protein intrinsic tryptophan (λ_ex 295 nm, λ_em 340 nm) and monitoring fluorescence from MANT (λ_ex 355 nm, λ_em 448 nm). 700 nM MANT-GTP was added and relative increase in fluorescence was monitored as a function of time. At 6000 seconds, 10 μM GTPγS was added to the reaction mixture. The fluorescence remaining after addition of GTPγS was subtracted from the data. Non-covalent interaction resulted in retardation in basal Gα_i1 mGTP uptake in a AuNP concentration dependent manner.