Early-Onset Hypertrophic Cardiomyopathy Mutations Significantly Increase the Velocity, Force, and Actin-Activated ATPase Activity of Human ?-Cardiac Myosin.
2016; 17 (11): 2857?64
Conformational Dynamics Accompanying the Proteolytic Degradation of Trimeric Collagen I by Collagenases
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2012; 134 (32): 13259-13265
Hypertrophic cardiomyopathy (HCM) is a heritable cardiovascular disorder that affects 1 in 500 people. A significant percentage of HCM is attributed to mutations in ?-cardiac myosin, the motor protein that powers ventricular contraction. This study reports how two early-onset HCM mutations, D239N and H251N, affect the molecular biomechanics of human ?-cardiac myosin. We observed significant increases (20%-90%) in actin gliding velocity, intrinsic force, and ATPase activity in comparison to wild-type myosin. Moreover, for H251N, we found significantly lower binding affinity between the S1 and S2 domains of myosin, suggesting that this mutation may further increase hyper-contractility by releasing active motors. Unlike previous HCM mutations studied at the molecular level using human ?-cardiac myosin, early-onset HCM mutations lead to significantly larger changes in the fundamental biomechanical parameters and show clear hyper-contractility.
View details for DOI 10.1016/j.celrep.2016.11.040
View details for PubMedID 27974200
Mechanical Load Induces a 100-Fold Increase in the Rate of Collagen Proteolysis by MMP-1
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2011; 133 (6): 1686-1689
Collagenases are the principal enzymes responsible for the degradation of collagens during embryonic development, wound healing, and cancer metastasis. However, the mechanism by which these enzymes disrupt the highly chemically and structurally stable collagen triple helix remains incompletely understood. We used a single-molecule magnetic tweezers assay to characterize the cleavage of heterotrimeric collagen I by both the human collagenase matrix metalloproteinase-1 (MMP-1) and collagenase from Clostridium histolyticum. We observe that the application of 16 pN of force causes an 8-fold increase in collagen proteolysis rates by MMP-1 but does not affect cleavage rates by Clostridium collagenase. Quantitative analysis of these data allows us to infer the structural changes in collagen associated with proteolytic cleavage by both enzymes. Our data support a model in which MMP-1 cuts a transient, stretched conformation of its recognition site. In contrast, our findings suggest that Clostridium collagenase is able to cleave the fully wound collagen triple helix, accounting for its lack of force sensitivity and low sequence specificity. We observe that the cleavage of heterotrimeric collagen is less force sensitive than the proteolysis of a homotrimeric collagen model peptide, consistent with studies suggesting that the MMP-1 recognition site in heterotrimeric collagen I is partially unwound at equilibrium.
View details for DOI 10.1021/ja212170b
View details for Web of Science ID 000307487200030
View details for PubMedID 22720833
Effects of hypertrophic and dilated cardiomyopathy mutations on power output by human beta-cardiac myosin
JOURNAL OF EXPERIMENTAL BIOLOGY
2016; 219 (2): 161-167
Mechanical coordination in motor ensembles revealed using engineered artificial myosin filaments
Strain Tunes Proteolytic Degradation and Diffusive Transport in Fibrin Networks
2012; 13 (2): 499-506
Although mechanical stress is known to profoundly influence the composition and structure of the extracellular matrix (ECM), the mechanisms by which this regulation occurs remain poorly understood. We used a single-molecule magnetic tweezers assay to study the effect of force on collagen proteolysis by matrix metalloproteinase-1 (MMP-1). Here we show that the application of ?10 pN in extensional force causes an ?100-fold increase in proteolysis rates. Our results support a mechanistic model in which the collagen triple helix unwinds prior to proteolysis. The data and resulting model predict that biologically relevant forces may increase localized ECM proteolysis, suggesting a possible role for mechanical force in the regulation of ECM remodeling.
View details for DOI 10.1021/ja109972p
View details for Web of Science ID 000287831800020
View details for PubMedID 21247159
Multiplexed single-molecule force proteolysis measurements using magnetic tweezers.
Journal of visualized experiments : JoVE
Proteolytic degradation of fibrin, the major structural component in blood clots, is critical both during normal wound healing and in the treatment of ischemic stroke and myocardial infarction. Fibrin-containing clots experience substantial strain due to platelet contraction, fluid shear, and mechanical stress at the wound site. However, little is understood about how mechanical forces may influence fibrin dissolution. We used video microscopy to image strained fibrin clots as they were degraded by plasmin, a major fibrinolytic enzyme. Applied strain causes up to 10-fold reduction in the rate of fibrin degradation. Analysis of our data supports a quantitative model in which the decrease in fibrin proteolysis rates with strain stems from slower transport of plasmin into the clot. We performed fluorescence recovery after photobleaching (FRAP) measurements to further probe the effect of strain on diffusive transport. We find that diffusivity perpendicular to the strain axis decreases with increasing strain, while diffusivity along the strain axis remains unchanged. Our results suggest that the properties of the fibrin network have evolved to protect mechanically loaded fibrin from degradation, consistent with its function in wound healing. The pronounced effect of strain upon diffusivity and proteolytic susceptibility within fibrin networks offers a potentially useful means of guiding cell growth and morphology in fibrin-based biomaterials.
View details for DOI 10.1021/bm2015619
View details for Web of Science ID 000300115900025
View details for PubMedID 22185486
pH-Dependent Formation of Lipid Heterogeneities Controls Surface Topography and Binding Reactivity in Functionalized Bilayers
2009; 25 (14): 8144-8151
The generation and detection of mechanical forces is a ubiquitous aspect of cell physiology, with direct relevance to cancer metastasis(1), atherogenesis(2) and wound healing(3). In each of these examples, cells both exert force on their surroundings and simultaneously enzymatically remodel the extracellular matrix (ECM). The effect of forces on ECM has thus become an area of considerable interest due to its likely biological and medical importance(4-7). Single molecule techniques such as optical trapping(8), atomic force microscopy(9), and magnetic tweezers(10,11) allow researchers to probe the function of enzymes at a molecular level by exerting forces on individual proteins. Of these techniques, magnetic tweezers (MT) are notable for their low cost and high throughput. MT exert forces in the range of ~1-100 pN and can provide millisecond temporal resolution, qualities that are well matched to the study of enzyme mechanism at the single-molecule level(12). Here we report a highly parallelizable MT assay to study the effect of force on the proteolysis of single protein molecules. We present the specific example of the proteolysis of a trimeric collagen peptide by matrix metalloproteinase 1 (MMP-1); however, this assay can be easily adapted to study other substrates and proteases.
View details for DOI 10.3791/3520
View details for PubMedID 22871786
During direct cell-to-cell communication, lipids on the extracellular side of plasma membranes reorganize, and membrane-associated communication-related molecules colocalize. At colocalization sites, sometimes identified as rafts, the local cell surface topography and reactivity are altered. The processes regulating these changes are largely unknown. On model lipid membranes, study of simplified processes that control surface topography and reactivity may potentially contribute to the understanding and control of related cell functions and associated diseases. Integration of these processes on nanometer-sized lipid vesicles used as drug delivery carriers would precisely control their interactions with diseased cells minimizing toxicities. Here we design such basic pH-dependent processes on model functionalized lipid bilayers, and we demonstrate reversible sharp changes in binding reactivity within a narrow pH window. Cholesterol enables tuning of the membrane reorganization to occur at pH values not necessarily close to the reported pK(a)'s of the constituent titratable lipids, and bilayer reorganization over repeated cycles of induced pH changes exhibits hysteresis.
View details for DOI 10.1021/la9004032
View details for Web of Science ID 000268138400049
View details for PubMedID 19594187