Inhibition of chymotrypsin through surface binding using nanoparticle-based receptors

Abstract

Efficient binding of biomacromolecular surfaces by synthetic systems requires the effective presentation of complementary elements over large surface areas. We demonstrate here the use of mixed monolayer protected gold clusters (MMPCs) as scaffolds for the binding and inhibition of chymotrypsin. In these studies anionically functionalized amphiphilic MMPCs were shown to inhibit chymotrypsin through a two-stage mechanism featuring fast reversible inhibition followed by a slower irreversible process. This interaction is very efficient, with a K(i)(app) = 10.4 +/- 1.3 nM. The MMPC-protein complex was characterized by CD, demonstrating an almost complete denaturation of the enzyme over time. Dynamic light scattering studies confirm that inhibition proceeds without substantial MMPC aggregation. The electrostatic nature of the engineered interactions provides a level of selectivity: little or no inhibition of function was observed with elastase, beta-galactosidase, or cellular retinoic acid binding protein.

Figure 1

Fabrication of MMPCs by using the Brust reduction and the Murray place-displacement reaction to introduce additional functionalized thiols.

Figure 2

(a) Space-filling model of ChT. Surface binding of the protein by anionic MMPCs focuses on the ring of cationic residues situated around the active site. Functionally significant residues are noted. (b) Relative sizes of ChT and MMPC 1.

Figure 3

Anionic MMPCs 1 and 2 and cationic control 3.

Figure 4

Time course of ChT activity after preincubation with MMPCs ([ChT] = 800 nM). (a) Anionic nanoparticles 1 and 2 and cationic control MMPC 3. (b) Concentration dependence of MMPC 1 inhibition. Lines are provided to lead the eye.

Figure 5

CD of ChT ([ChT] = 3.2 μM). (a) After initial mixing. (b) After 24-h incubation and thermal denaturation (green trace).

Figure 6

Kinetic analysis of MMPC 1 inhibition. (a) Progress curves of ChT activity illustrating the decrease in activity and product formation over time as MMPC 1 concentration is increased. Concentration of ChT was fixed at 10 nM. Chromogenic substrate N-Suc-AAPF-pNA concentration was kept at 150 μM. MMPC 1 concentrations were 1, 2, 5, and 10 nM. (b) Plot of k_obs vs. MMPC 1 concentration, from which K was calculated by nonlinear curve fitting. Calculations were completed by assuming a 1:5 nanoparticle/ChT binding ratio.

Figure 7

Reciprocal plot of 1/k_obs vs. 1/[MMPC 1], showing nonzero intercept indicative of a two-step inactivation mechanism.