Estimation of microscope drift using fluorescent nanodiamonds as fiducial markers

Nash

et al. 2020

Biophysical Journal

An understanding of the structure-dynamics relationship is essential for understanding how a protein works. Prior research has shown that the activity of a protein correlates with intradomain dynamics occurring at picosecond to millisecond timescales. However, the correlation between interdomain dynamics and the function of a protein is poorly understood. Here, we show that communications between the catalytic and hemopexin domains of matrix metalloprotease-1 (MMP1) on type 1 collagen fibrils correlate with its activity. Using single-molecule Förster resonance energy transfer, we identified functionally relevant open conformations in which the two MMP1 domains are well separated, which were significantly absent for catalytically inactive point mutant (E219Q) of MMP1 and could be modulated by an inhibitor or an enhancer of activity. The observed relevance of open conformations resolves the debate about the roles of open and closed MMP1 structures in function. We fitted the histograms of single-molecule Förster resonance energy transfer values to a sum of two Gaussians and the autocorrelations to an exponential and power law. We used a two-state Poisson process to describe the dynamics and calculate the kinetic rates from the fit parameters. All-atom and coarse-grained simulations reproduced some of the experimental features and revealed substrate-dependent MMP1 dynamics. Our results suggest that an interdomain separation facilitates opening up the catalytic pocket so that the collagen chains come closer to the MMP1 active site. Coordination of functional conformations at different parts of MMP1 occurs via allosteric communications that can take place via interactions mediated by collagen even if the linker between the domains is absent. Modeling dynamics as a Poisson process enables connecting the picosecond timescales of molecular dynamics simulations with the millisecond timescales of single-molecule measurements. Water-soluble MMP1 interacting with water-insoluble collagen fibrils poses challenges for biochemical studies that the single-molecule tracking can overcome for other insoluble substrates. Interdomain communications are likely important for multidomain proteins.

Section: Resultsmentioning

confidence: 99%

Allosteric Communications between Domains Modulate the Activity of Matrix Metalloprotease-1

Nash

et al. 2020

Biophysical Journal

“…The quartz slides had two holes to connect the input and output tubes for exchanging buffers and solutions. We created an evanescent wave at the slide's interface in a Total Internal Reflection Fluorescence (TIRF) Microscope, as described before 39,40,65,66 . We incubated 50 µL of 0.1 mg/mL labeled MMP1 with (1) 50 µL of protein buffer (50 mM Tris, 100 mM NaCl, pH 8.0), (2) 50 µL of 1 mg/mL MMP9, and (3) 50 µL of 0.1 mg/mL tetracycline for 30 min at 22 °C.…”

Section: Unlike Collagen Mmp1 Domains Do Not Communicate Via Fibrinmentioning

confidence: 99%

Activity-dependent interdomain dynamics of matrix metalloprotease-1 on fibrin

Planas-Iglesias

et al. 2020

Sci Rep

The roles of protein conformational dynamics and allostery in function are well-known. However, the roles that interdomain dynamics have in function are not entirely understood. We used matrix metalloprotease-1 (MMP1) as a model system to study the relationship between interdomain dynamics and activity because MMP1 has diverse substrates. Here we focus on fibrin, the primary component of a blood clot. Water-soluble fibrinogen, following cleavage by thrombin, self-polymerize to form water-insoluble fibrin. We studied the interdomain dynamics of MMP1 on fibrin without crosslinks using single-molecule Forster Resonance Energy Transfer (smFRET). We observed that the distance between the catalytic and hemopexin domains of MMP1 increases or decreases as the MMP1 activity increases or decreases, respectively. We modulated the activity using (1) an active site mutant (E219Q) of MMP1, (2) MMP9, another member of the MMP family that increases the activity of MMP1, and (3) tetracycline, an inhibitor of MMP1. We fitted the histograms of smFRET values to a sum of two Gaussians and the autocorrelations to an exponential and power law. We modeled the dynamics as a two-state Poisson process and calculated the kinetic rates from the histograms and autocorrelations. Activity-dependent interdomain dynamics may enable allosteric control of the MMP1 function.

“…Reflection Fluorescence (TIRF) Microscope as described before [65][66][67][68] . We incubated 50 µL of 0.1 399 mg/mL labeled MMP1 with 50 µL of protein buffer (50 mM Tris, mM NaCl, pH 8.0) and 100 400 µg/mL tetracycline for 30 min at 22 C. The labeled MMP1 was serially diluted to prepare a 401 working concentration of ~100 pM and flowed into the flow cell.…”

Section: Introductionmentioning

confidence: 99%

Inter-domain dynamics and fibrinolytic activity of matrix metalloprotease-1

Planas-Iglesias

et al. 2019

Preprint

AbstractThe roles of protein conformational dynamics and allostery in function are well-known. However, the roles that inter-domain dynamics have in function are not entirely understood. We used matrix metalloprotease-1 (MMP1) as a model system to study the relationship between inter-domain dynamics and activity because MMP1 has diverse substrates. Here we focus on fibrin, the primary component of a blood clot. Water-soluble fibrinogen, following cleavage by thrombin, self-polymerize to form water-insoluble fibrin. We studied the inter-domain dynamics of MMP1 on fibrin without crosslinks using single-molecule Forster Resonance Energy Transfer (smFRET). We observed that the distance between the catalytic and hemopexin domains of MMP1 increases or decreases as the MMP1 activity increases or decreases, respectively. We modulated the activity using 1) an active site mutant (E219Q) of MMP1, 2) MMP9, another member of the MMP family that increases the activity of MMP1, and 3) tetracycline, an inhibitor of MMP1. We fitted the histograms of smFRET values to a sum of two Gaussians and the autocorrelations to an exponential and power law. We modeled the dynamics as a two-state Poisson process and calculated the kinetic rates from the histograms and autocorrelations. Activity-dependent inter-domain dynamics may enable allosteric control of the MMP1 function.