Loading-device effects on the protein-unfolding mechanisms using molecular-dynamic simulations

Highlights

• This study shows the unfolding of ubiquitin with two different loading devices.

• We confirmed the anisotropic unfolding of the protein due to rotation of α-helix.

• Anisotropic unfolding can confirm with geometrical and hydrogen bond analyses.

Abstract

Experimental force spectroscopy has been effectively utilized for measuring structural characterization of biomolecules and mechanical properties of biomaterials. Specifically, atomic force microscopy (AFM) has been widely used to portray biomolecular characterization in single-molecule experiment by observing the unfolding behavior of the proteins. Not only the experimental techniques enable us to characterize globular protein, but computational methods like molecular dynamics (MD) also gives insight into understanding biomolecular structures. To better comprehend the behavior of biomolecules, conditions such as pulling velocities and loading rates are put to the test, yet there are still limitations in understanding the unfolding behavior of biomolecules with the effect of different loading devices. In this study, we performed an all-atom MD and steered molecular dynamics (SMD) simulations considering different loading device effects such as “soft” and “stiff” to characterize the anisotropic unfolding behavior of ubiquitin protein. We found out the anisotropic unfolding pathways of the protein through the broken number of hydrogen bonds and geometric secondary structures of the biomolecule. Our study provides the importance for usage of various loading-devices on biomolecules when analyzing the structural compositions and the characteristics of globular biomolecules.

Introduction

Single-molecule experiment techniques have been efficiently used for the structural characterization of biomolecules like DNA, RNA, and globular proteins. Experimental force spectroscopies such as atomic force microscopy (AFM), magnetic tweezer and optical tweezer provide the biomolecular motion and the functional features by capturing the biomolecular unfolding behavior and measuring its rupture forces [1]. The principle of the single-molecule manipulation is through a force measurement between the target biomolecules connected to the loading device and the remaining molecules covalently attaching to substrates [[2], [3], [4]]. Not only are the single-molecule experiments applied to biomolecular characterizations, but they are also used to measure the topology compositions and the mechanical characteristics of biomaterials such as silks [[5], [6], [7]], collagen [8], ankyrin [9], actin [10], and intrinsically disordered proteins (IDPs) [[11], [12], [13], [14]].

Amid the biomolecules, ubiquitin protein has been typically studied for biomolecular characterization via the experimental-force-spectroscopy methods [4,15,16]. For example, Carrion-Vazquez et al. investigated the mechanical stability of the ubiquitin protein with different linkages [4]. They pulled the different sites (i.e., N-C and Lys48-C) of the protein and found different rupture forces and force-extension patterns after the AFM experiment. On the basis of the experimental analysis, theoretical analyses have supported the experimental studies of the ubiquitin-unfolding pathways in terms of various pulling-site aspects [15]. For example, Li et al. provided the different ubiquitin-unfolding linkage effects using the all-atom molecular dynamics (MD) and steered molecular dynamics (SMD) techniques [15]. Not only did they supported the unfolding trajectories that were experimentally revealed by Carrion-Vasquez et al. [4], but they also analyzed the way the different pulling sites of the lysine residues (i.e., Lys11, and Lys 64) alter the unfolding trajectories of the ubiquitin protein. On the contrary to the technique of Li et al. [15], Cieplak et al. characterized the structural feature of ubiquitin and the polyubiquitin structures using a coarse-grained (CG) method and the “Go-model” theory via a comparison of the previously reported experiment results [16]. Through the force-extension and force-distance of ubiquitin-unfolding simulation, they provided the similar ubiquitin-unfolding mechanism compared with the experiment results despite the application of the CG model and the Go model. Specifically, the sawtooth patterns explain the structural and conformational characterization of polyubiquitin through force-extension curves. Also, Cieplak et al. identified the ways that the ubiquitin motion and the unfolding mechanism changes when they applied various loading-speed conditions [16]. In addition to the ubiquitin studies, experimental and theoretical single-molecule techniques have been applied to other biomolecules using mechanistic analyses.

For the structural characterization of the biomolecule and the mechanistic analysis under a fast transition status, some experimental and theoretical researchers varied the external-force conditions such as the loading-velocity and device-loading rates. Since forces ranging from micro-to nanonewtons can elucidate complex biomolecular characterizations and their unfolding mechanisms, Carrion-Vasquez et al. experimentally and computationally investigated the unfolding pathway and the mechanical-stability measurements (i.e., the unfolding force) of ubiquitin via different loading-velocity conditions [4]. From the deep knowledge of the theoretical efforts, Maitra and Arya reported on the relationship between a wide range of loading-condition variations (i.e., the pulling-rate and pulling-device stiffness values) and the specific molecular-geometry reactions (i.e., contour length and persistence length) based on Kramers' theory [17]. Similarly, Yoon et al. also performed an ubiquitin-unfolding simulation with variations of the loading rate and the loading devices based on Brownian dynamics and the CG technique [18]. They revealed the anisotropic unfolding pathways and the protein reaction forces upon the application of the external-force condition. Despite the application of various loading rates and devices that are based on the CG technique and Brownian dynamics, different rupture forces emerged along with a rough anisotropic protein behavior with respect to the loading device and rates, but not the detailed unfolding trajectories. Recently, all-atom MD and SMD provided details of the biomolecular unfolding mechanism and the biomolecular mechanical-property measurement. Moreover, the structural and mechanical characterizations of the crystalline proteins were performed with the variation of the loading-velocity conditions. For example, Choi et al. and Lee et al., analyzed the mechanical properties and the bending behavior of human-islet-polypeptide (hIAPP) and transthyretin (TTR) fibrils using SMD simulations with variations of the various loading-device velocities [19,20]. Based on an understanding that the crystalline hIAPP fibrils are composed of rich β-sheets, the anisotropic behaviors of the fibrils are nonexistent during the varying of the loading-velocity conditions. Some computational study represented the conditions for loading device variation on the biomolecules including the crystalline protein structures using the MD and the SMD [24], but lack of the detailed unfolding trajectories of the anisotropic globular biomolecules that persisted in consideration of the various loading-device conditions became evident.

In the present study, we performed different loading-device effects on the globular biomolecule using all-atom MD and SMD simulations. The N-C pulling simulations on the ubiquitin were performed with various loading-device conditions such as 100, 200, 300, and 500 pN/Å, respectively. Compared with the previous ubiquitin-unfolding CG simulation for which the Brownian-dynamics theory was considered, we provided detailed aspect of protein-unfolding pathways upon the application of the two distinct spring constants of 100 pN/Å and 500 pN/Å. In addition, two distinct ubiquitin-unfolding pathways were observed through the conformational variations of secondary structures (i.e. α-helix and β-strands), the broken number of hydrogen bonds, and its geometrical analyses. The present study might provide a useful measurement for the unfolding simulation and the structural characterization of a globular protein via the application of the proper loading devices.