Figure 2

Time course AlF^4-mediated Gα _i1 activation of bioconjugated AuNP- Gα _i1 . (A) Demonstrates effect of non-covalent interaction of AuNP- Gα_i1 on rate of AlF^4- binding to Gα_i1-GDP. Solid line represents 0 nM AuNP (only protein); Short dash-short dash line represents 0.1 nM AuNP; dotted line represents 0.5 nM AuNP; dashed line represents 0.75 nM AuNP; dash-dot-dot line represents 1 nM AuNP. (B) Demonstrates effect of N-terminal covalent conjugation of Gα_i1 to AuNP via EDC reaction on AlF^4- binding to AuNP- Gα_i1-GDP. The solid line represents Gα_i1 without any conjugation (control); the dash-dot-dot line represents conjugated AuNP- Gα_i1. For all the time course fluorescence measurement final concentration of 200 nM of Gα_i1 (conjugated and purified 200 nM AuNP- Gα_i1 in case of covalent conjugation) was taken in a quartz cuvette containing 5 mM Hepes-Na (pH 8.0), 10 mM NaCl, 0.5 mM MgCl₂, 1 μM GDP. In case of experiments performed in panel A, appropriate amounts of AuNP-DHLA (in 5 mM Hepes-Na (pH 8.0)) were mixed with protein and incubated for 10 minutes. Tryptophan emission at 340 nm was monitored by exciting the sample at 295 nm with continuous stirring. 2 mM NaF and subsequently followed by 20 μM AlCl₃ was added to the reaction and relative fluorescence was monitored as a function of time. All the measurements were performed at 25°C. Non-covalently conjugated AuNP- Gα_i1 displayed deaccelerated rates of basal AlF^4- binding to Gα_i1-GDP. Non covalent conjugates decreased AlF^4- binding upto 0.08 fold. On the contrary N-terminal covalent conjugation caused 3.2 fold increase in rate of AlF^4- binding. The Plateau fluorescence intensity of covalently conjugated AuNP- Gα_i1 was comparable to only Gαi1, whereas non-covalent conjugation displayed decrease in plateau fluorescence in a concentration dependent manner.