Conformational distribution of protein molecules revealed with multi-tilt nanoparticle-aided cryo-electron microscopy sampling

Abstract

Understanding the conformational distribution of protein molecules is essential for comprehending their biological functions. Conventional structural determination techniques typically provide the average ensemble features, with limitations in measuring the heterogeneous conformational distributions within individual protein molecules. Especially, small-sized proteins are known to participate in biological responses by exhibiting a wide range of dynamic, and heterogeneous conformations rather than a single equilibrium structure. Therefore, to understand the functions of small proteins, experimental methods are required that can statistically visualize the heterogeneous conformational distributions that proteins can adopt. Here, we present an experimental method, "Multi-Tilt Nanoparticle-Aided Cryo-electron microscopy Sampling (MT-NACS)” to determine the conformational distribution of calmodulin (CaM) protein upon ligand binding and variations of salt concentrations. Due to the difficulty in obtaining enough contrast in cryo-electron microscopy images for small protein molecule, gold nanoparticles were utilized as contrast agents. By imaging CaM proteins labeled by two gold nanoparticles at multiple tilt angles and analyzing the projected positions of gold nanoparticles, we can extract the three-dimensional interparticle distance between two gold nanoparticles of CaM. By collecting sufficient images of gold nanoparticle-labeled CaM, the distributions of 3D interparticle distances of CaM were obtained. First, we confirmed the feasibility of our method by comparing the results of MT-NACS experiments, which investigated structural changes induced by calcium ion binding and variations in salt concentration, with previous studies. Additionally, MT-NACS was utilized to track the structural changes upon binding of amyloid beta peptide to CaM, which has never been observed experimentally. We anticipate that this experimental work will offer an alternative platform for unraveling the functional flexibility of small protein molecules.