Atomic Force Microscopy Study of the Conformational Change in Immobilized Calmodulin

Abstract

Maintaining the biological functionality of immobilized proteins is the key to the success of numerous protein-based biomedical devices. To that end, we studied conformational change of calmodulin (CaM) immobilized on chemical patterns. 1-cysteine mutated calmodulin was immobilized on a mercapto-terminated surface through the cysteine-Hg-mercapto coupling. Utilizing Atomic Force Microscope (AFM), the average height of the immobilized calmodulin was determined to be 1.87 ± 0.19 nm. After incubation in EGTA solution, the average height of protein changed to 2.26 ± 0.21 nm, indicating conformational change of CaM to Apo-CaM. The immobilized CaM also demonstrated conformational change upon the reaction with known calmodulin antagonist chlorpromazine (CPZ). After incubation in CPZ solution, the average height of CPZ-bound CaM increased to 2.32 ± 0.20 nm, demonstrating the immobilized CaM still has the similar response as in bulk solution. These results show that immobilization of calmodulin on a solid support does not interfere with the ability of the protein to bind calcium and calmodulin antagonists. Our results demonstrate the feasibility of employing AFM to probe and understand protein conformational changes.

SP6616 as a new Kv2.1 channel inhibitor efficiently promotes β-cell survival involving both PKC/Erk1/2 and CaM/PI3K/Akt signaling pathways

“…Given that cytosolic-free calcium activates PI3K/Akt pathway through regulation of CaM 24 , 25 and SP6616-induced Ca 2+ influx, we next investigated whether CaM stimulation linked Kv2.1 inhibition to PI3K/Akt pathway activation in INS-832/13 cells. As indicated in Figures 4o and p , incubation of CaM antagonist chlorpromazine (CPZ) 26 caused almost the inactivity of SP6616 in reversing the STZ-reduced Akt phosphorylation.…”

Investigating and characterizing the binding activity of the immobilized calmodulin to calmodulin-dependent protein kinase I binding domain with atomic force microscopy

“…5 μg mL −1 CaM with buffer solution (25 mmol L −1 Tris–HCl, 1 mmol L −1 CaCl 2 , pH 8.0) was deposited onto the pattern area for one hour in refrigerator at 4 °C (Fig. 3 d) [ 32 ]. Then the sample surface was wiped with a piece of ChemWipe paper, in a typical force of 1 N [ 33 ], to remove the nonspecifically adsorbed protein on the OTS background, while those specifically bind to substrate surface remained.…”

A Method for Attaching Thiol Groups Directly on a Silicon (111) Substrate

“…Silicon wafers were cut into 1 cm × 1 cm pieces. The wafer samples were cleaned by piranha solution (1 part of H2SO4 and 2 parts of H2O2) at 125°C for 15 min 19,20 . After rinsing the samples in deionized water and drying under a stream of N2(g), the oxidized silicon samples were etched in the 5 : 1 (V/V) NH4F/HF(aq) solution for 1 min and then, without rinsing, were immersed into 40% NH4F(aq) solution for 10 min 21 .…”