Jongmin Sung

Molecular consequences of the R453C hypertrophic cardiomyopathy mutation on human β-cardiac myosin motor function.

Cardiovascular disorders are the leading cause of morbidity and mortality in the developed world, and hypertrophic cardiomyopathy (HCM) is among the most frequently occurring inherited cardiac disorders. HCM is caused by mutations in the genes encoding the fundamental force-generating machinery of the cardiac muscle, including β-cardiac myosin. Here, we present a biomechanical analysis of the HCM-causing mutation, R453C, in the context of human β-cardiac myosin. We found that this mutation causes a ∼30% decrease in the maximum ATPase of the human β-cardiac subfragment 1, the motor domain of myosin, and a similar percent decrease in the in vitro velocity. The major change in the R453C human β-cardiac subfragment 1 is a 50% increase in the intrinsic force of the motor compared with wild type, with no appreciable change in the stroke size, as observed with a dual-beam optical trap. These results predict that the overall force of the ensemble of myosin molecules in the muscle should be higher in the R453C mutant compared with wild type. Loaded in vitro motility assay confirms that the net force in the ensemble is indeed increased. Overall, this study suggests that the R453C mutation should result in a hypercontractile state in the heart muscle.

Combining Single-Molecule Optical Trapping and Small-Angle X-Ray Scattering Measurements to Compute the Persistence Length of a Protein ER/K α-Helix

A relatively unknown protein structure motif forms stable isolated single alpha-helices, termed ER/K alpha-helices, in a wide variety of proteins and has been shown to be essential for the function of some molecular motors. The flexibility of the ER/K alpha-helix determines whether it behaves as a force transducer, rigid spacer, or flexible linker in proteins. In this study, we quantify this flexibility in terms of persistence length, namely the length scale over which it is rigid. We use single-molecule optical trapping and small-angle x-ray scattering, combined with Monte Carlo simulations to demonstrate that the Kelch ER/K alpha-helix behaves as a wormlike chain with a persistence length of approximately 15 nm or approximately 28 turns of alpha-helix. The ER/K alpha-helix length in proteins varies from 3 to 60 nm, with a median length of approximately 5 nm. Knowledge of its persistence length enables us to define its function as a rigid spacer in a translation initiation factor, as a force transducer in the mechanoenzyme myosin VI, and as a flexible spacer in the Kelch-motif-containing protein.

Single-Molecule Dual-Beam Optical Trap Analysis of Protein Structure and Function

Optical trapping is one of the most powerful single-molecule techniques. We provide a practical guide to set up and use an optical trap, applied to the molecular motor myosin as an example. We focus primarily on studies of myosin function using a dual-beam optical trap, a protocol to build such a trap, and the experimental and data analysis protocols to utilize it.

Effects of hypertrophic and dilated cardiomyopathy mutations on power output by human β-cardiac myosin.

ABSTRACT Hypertrophic cardiomyopathy is the most frequently occurring inherited cardiovascular disease, with a prevalence of more than one in 500 individuals worldwide. Genetically acquired dilated cardiomyopathy is a related disease that is less prevalent. Both are caused by mutations in the genes encoding the fundamental force-generating protein machinery of the cardiac muscle sarcomere, including human β-cardiac myosin, the motor protein that powers ventricular contraction. Despite numerous studies, most performed with non-human or non-cardiac myosin, there is no clear consensus about the mechanism of action of these mutations on the function of human β-cardiac myosin. We are using a recombinantly expressed human β-cardiac myosin motor domain along with conventional and new methodologies to characterize the forces and velocities of the mutant myosins compared with wild type. Our studies are extending beyond myosin interactions with pure actin filaments to include the interaction of myosin with regulated actin filaments containing tropomyosin and troponin, the roles of regulatory light chain phosphorylation on the functions of the system, and the possible roles of myosin binding protein-C and titin, important regulatory components of both cardiac and skeletal muscles. Summary: The underlying molecular basis of genetic-based cardiomyopathy diseases is largely unknown. This review describes recent molecular studies that have used human cardiac proteins to begin to elucidate the mechanisms whereby mutations cause disease.

Observation of correlated X-ray scattering at atomic resolution.

Tools to study disordered systems with local structural order, such as proteins in solution, remain limited. Such understanding is essential for e.g. rational drug design. Correlated X-ray scattering (CXS) has recently attracted new interest as a way to leverage next-generation light sources to study such disordered matter. The CXS experiment measures angular correlations of the intensity caused by the scattering of X-rays from an ensemble of identical particles, with disordered orientation and position. Averaging over 15 496 snapshot images obtained by exposing a sample of silver nanoparticles in solution to a micro-focused synchrotron radiation beam, we report on experimental efforts to obtain CXS signal from an ensemble in three dimensions. A correlation function was measured at wide angles corresponding to atomic resolution that matches theoretical predictions. These preliminary results suggest that other CXS experiments on disordered ensembles—such as proteins in solution—may be feasible in the future.

Optimized measurements of separations and angles between intra-molecular fluorescent markers

We demonstrate a novel, yet simple tool for the study of structure and function of biomolecules by extending two-colour co-localization microscopy to fluorescent molecules with fixed orientations and in intra-molecular proximity. From each colour-separated microscope image in a time-lapse movie and using only simple means, we simultaneously determine both the relative (x,y)-separation of the fluorophores and their individual orientations in space with accuracy and precision. The positions and orientations of two domains of the same molecule are thus time-resolved. Using short double-stranded DNA molecules internally labelled with two fixed fluorophores, we demonstrate the accuracy and precision of our method using the known structure of double-stranded DNA as a benchmark, resolve 10-base-pair differences in fluorophore separations, and determine the unique 3D orientation of each DNA molecule, thereby establishing short, double-labelled DNA molecules as probes of 3D orientation of anything to which one can attach them firmly.

How to measure separations and angles between intra-molecular fluorescent markers

Structure and function of an individual biomolecule can be explored with minimum two fluorescent markers of different colors. Since the light of such markers can be spectrally separated and imaged simultaneously, the markers can be colocalized. Here, we describe the method used for such two-color colocalization microscopy. Then we extend it to fluorescent markers with fixed orientations and in intramolecular proximity. Our benchmarking of this extension produced two extra results: (a) we established short double-labeled DNA molecules as probes of 3D orientation of anything to which one can attach them firmly; (b) we established how to map with super-resolution between color-separated channels, which should be useful for all dual-color colocalization measurements with either fixed or freely rotating fluorescent molecules. Throughout, we use only simple means: from each color-separated microscope image in a time-lapse movie, we simultaneously determine both the relative (x,y)-separation of the fluorophores and their individual orientations in space, both with accuracy and precision. The relative positions and orientations of two domains of the same molecule are thus time-resolved. Using short double-stranded DNA (dsDNA) molecules internally labeled with two fixed fluorophores, we (i) demonstrate the accuracy and precision of our localization- and mapping-methods, using the known structure of dsDNA as benchmark; (ii) resolve 10 base pair differences in fluorophore separations; (iii) determine the unique 3D orientation of each DNA molecule.

Chapter Six – How to Measure Separations and Angles Between Intramolecular Fluorescent Markers

Structure and function of an individual biomolecule can be explored with minimum two fluorescent markers of different colors. Since the light of such markers can be spectrally separated and imaged simultaneously, the markers can be colocalized. Here, we describe the method used for such two-color colocalization microscopy. Then we extend it to fluorescent markers with fixed orientations and in intramolecular proximity. Our benchmarking of this extension produced two extra results: (a) we established short double-labeled DNA molecules as probes of 3D orientation of anything to which one can attach them firmly; (b) we established how to map with super-resolution between color-separated channels, which should be useful for all dual-color colocalization measurements with either fixed or freely rotating fluorescent molecules. Throughout, we use only simple means: from each color-separated microscope image in a time-lapse movie, we simultaneously determine both the relative (x,y)-separation of the fluorophores and their individual orientations in space, both with accuracy and precision. The relative positions and orientations of two domains of the same molecule are thus time-resolved. Using short double-stranded DNA (dsDNA) molecules internally labeled with two fixed fluorophores, we (i) demonstrate the accuracy and precision of our localization- and mapping-methods, using the known structure of dsDNA as benchmark; (ii) resolve 10 base pair differences in fluorophore separations; (iii) determine the unique 3D orientation of each DNA molecule.

Understanding the Effects of Cardiomyopathy Causing Mutations on Human Beta Cardiac Myosin Biomechanical Function

Cardiomyopathy is one of the leading causes of sudden cardiac death across the developed world. Over the last twenty years numerous single point mutations in the cardiac contractile apparatus have been implicated in hypertrophic and dilated cardiomyopathy (HCM or DCM), out of which nearly 40% of them are housed in the human β-cardiac myosin. Nonetheless, the underlying molecular effects of most of these mutations remain elusive. The cardiac muscle cyclically contracts under varying loads and it is likely that the mutations in the β-cardiac myosin affect the power output of the heart. We hypothesize that these mutations cause a fundamental mechanistic change in the power output of the cardiac myosin contractile system by either altering force or velocity of contraction, which ultimately compromises heart function and results in the clinical phenotypes.Using a combination of biochemistry and single molecule spectroscopy we show that two HCM-causing mutations (R403Q and R453C) in a truncated version of human β-cardiac myosin ending after the essential light chain (1-808) cause an increase in the power output by two different mechanisms. For R403Q we see a ∼20% increase in the maximum unloaded velocity, whereas for R453C the major change is a ∼50% increase in the intrinsic force generated by a single myosin head. On the other hand, with the DCM-causing S532P β-cardiac myosin mutant both unloaded velocity and intrinsic force is decreased as compared to the wild type, predicting a lower power output. These results indicate that distinct molecular mechanisms for altering power output are operative for different cardiomyopathy-causing mutants. Work is currently being performed to understand these and other cardiomyopathy-causing mutations using an extended version of the human β-cardiac myosin molecule (1-843) with both human light chains.

Correlated scattering: probing atomic structure of molecules and nanoparticles

In 1977, Z. Kam theorized that correlations of scattering patterns, measured by exposing a solution of randomly oriented identical particles to x-ray radiation, could yield detailed information on the internal structure of the individual particles [1]. During a single exposure (whose duration should be short compared to the particle rotational diffusion time), physical correlations arise whenever multiple photons scatter from the same particle into different directions. By averaging correlations from many exposures, we have demonstrated that one can extract this correlated signal from a background of uncorrelated single-direction scattering events from different particles [2]. This additional information can be used to place constraints on model structures of the particles under investigation, providing a method of structure refinement to atomic resolution. We recently observed correlated scattering from solutions of ~10^9 silver nanoparticles exposed to synchrotron radiation at a microfocus beamline at SSRL [2]. By autoand crosscorrelating the Bragg rings 111 and 200, five correlation peaks were resolved corresponding to the structure and symmetry of silver’s reciprocal lattice. To transition from nanoparticles to biomolecule studies, we have performed several experiments at x-ray free electron laser centers (SLAC and SPring-8), and are working to refine analysis techniques.