Cutting-Edge Single-Molecule Technologies Unveil New Mechanics in Cellular Biochemistry

Abstract

Single-molecule technologies have expanded our ability to detect biological events individually, in contrast to ensemble biophysical technologies, where the result provides averaged information. Recent developments in atomic force microscopy have not only enabled us to distinguish the heterogeneous phenomena of individual molecules, but also allowed us to view up to the resolution of a single covalent bond. Similarly, optical tweezers, due to their versatility and precisions, have emerged as a potent technique to dissect a diverse range of complex biological processes, from nanomechanics of ClpXP protease–dependent degradation to force-dependent processivity of motor proteins. Despite the advantages of optical tweezers, the time scales used in this technology were inconsistent with physiological scenarios, which led to the development of magnetic tweezers, where proteins are covalently linked with the glass surface, which in turn increases the observation window of a single biomolecule from minutes to weeks. Unlike optical tweezers, magnetic tweezers use magnetic fields to impose torque, which makes them convenient for studying DNA topology and topoisomerase functioning. Using modified magnetic tweezers, researchers were able to discover the mechanical role of chaperones, which support their substrate proteins by pulling them during translocation and assist their native folding as a mechanical foldase. In this article, we provide a focused review of many of these new roles of single-molecule technologies, ranging from single bond breaking to complex chaperone machinery, along with the potential to design mechanomedicine, which would be a breakthrough in pharmacological interventions against many diseases. Expected final online publication date for the Annual Review of Biophysics, Volume 50 is May 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Single-molecule mechanical studies of chaperones and their clients

“…While single-molecule studies of some chaperones have also been covered in several previous reviews [18][19][20][21][22][23][24][25][26][27][28], this section provides a short description of mechanical interrogation strategies, instruments for force experiments, and our perspective on selected contributions in chaperone-client interactions that have been published since 2015. The subsections are divided into heat shock proteins, the study of the impact of chaperones on folding ribosomebound proteins, DsbA-client interactions, SNARE proteins with Mun/Munc chaperones, and chaperone-assisted folding of the membrane proteins.…”

Single-Molecule Optical Tweezers As a Tool for Delineating the Mechanisms of Protein-Processing Mechanoenzymes

“…Single-molecule measurements give complementary information to the ensemble measurements because they can measure anisotropic properties such as force, track molecular displacements, and highlight the stochasticity of various properties, resulting in the visualization of the heterogeneity in molecular properties of different biological motors . In addition, single-molecule assays can give us information on individual steps during a multistep mechanism and the trajectories of individual molecules during a time course that is usually masked in bulk ensemble measurements. , These methods could be divided into either monitoring methods, such as single-molecule fluorescence imaging, or manipulating methods, known as single-molecule force-spectroscopy (SMFS). SMFS involves the study of the properties of individual molecules by probing them with force in the form of tension and includes techniques such as atomic force microscopy (AFM), optical tweezers (OT), and magnetic tweezers (MT) that have been widely used to divulge information on the motor properties of enzymes, mechanical unfolding pathways of proteins, as well as stabilizing effects of ligands on proteins. , Among these, optical-tweezer-based assays have championed over others in understanding the mechanisms of molecular motors, with their ability to precisely measure subnanometer displacements with microsecond temporal resolution, applying stretching forces in the range from subpiconewtons to hundreds of piconewtons. , In addition to the technical advantage of spatiotemporal resolution and range of forces the optical tweezers can measure (Table ), it gives an advantage over other methods due to its ability to steer the beads onto which molecules are attached, facilitating increased interactions between molecular motors and their substrates in the solution for faster data collection.…”

“…Spatiotemporal resolutions and measurable force ranges of commonly used SMFS techniques are given in Table . ,,− …”

Direct Observation of the Mechanical Role of Bacterial Chaperones in Protein Folding

“…Protein folding under force is an important source of generating mechanical energy that harnesses different biological functions ranging from protein translation to degradation. − For example, in translation, protein synthesis occurs in a confined ribosomal tunnel under 7–11 pN force, while ClpX machinery generates 4–15 pN force to unfold proteins, followed by ClpP-dependent degradation. , Notably, molecular chaperones, well involved in various folding processes, − must interact with proteins under force. Recent force spectroscopy studies have nicely illustrated the molecular mechanisms of various chaperones, but most of them focused on the role of chaperones in the folding–misfolding aspects of the client proteins. ,, However, few studies reported emerging evidence that molecular chaperones could modulate the force transmission through their client polypeptide during different folding-associated processes, including during cotranslational folding at the ribosomal tunnel edge .…”