Efficient protease based purification of recombinant matrix metalloprotease-1 in E. coli

Kumar, Lokender; Colomb, Warren; Czerski, John; Cox, Christopher R.; Sarkar, Susanta K.

doi:10.1016/j.pep.2018.04.001

Cited by 12 publications

(26 citation statements)

References 64 publications

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“…We mutated SER142 on the catalytic domain and SER366 on the hemopexin domain to cysteines ( Figure 1A ). Note that the amino acid E219 13 is the same as E200 5 , which differs due to the first residue used for counting. The distance between the central carbon atoms of the two selected amino acids is ∼4.5 nm, which is similar to the Förster radius of the dye pair Alexa Fluor® 555 and Alexa Fluor® 647.…”

Section: Resultsmentioning

confidence: 99%

“…Full-length recombinant MMPs were expressed in E. coli and purified in activated form using a protease based purification method 13 . Purified MMP1 was labeled with AlexaFluor555 (donor dye) and AlexaFluor647 (acceptor dye) using maleimide chemistry.…”

Section: Methods Summarymentioning

confidence: 99%

“…After incubation, the sample was filtered three times using a 30 kDa cut-off Amicon filter to remove free dyes from the solution. The labeled MMP1 was analyzed using 12% SDS PAGE and the activity of labeled MMP1 was compared with the activity of unlabeled protein on the synthetic substrate, MCA-Lys-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH2, (R&D Systems, Cat# ES010) as described before 13 .…”

Section: Methods Summarymentioning

confidence: 99%

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Allosteric communications between domains modulate the activity of matrix metalloprotease-1

Kumar

Nash

Harms

et al. 2019

Preprint

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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 intra-domain dynamics occurring at picosecond to millisecond timescales. However, the correlation between inter-domain 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 FRET (smFRET), we identified functionally relevant open conformations where 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. A sum of two Gaussians fitted histograms, whereas an exponential fitted autocorrelations of smFRET values. We used a two-state Poisson process to describe the dynamics and used histograms and autocorrelations of conformations to calculate the kinetic rates between the two states. All-atom and coarse-grained simulations reproduced some of the experimental features and revealed substrate-dependent MMP1 dynamics. Our results suggest that an inter-domain 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. Inter-domain communications are likely important for multidomain proteins.Statement of SignificanceIt is often challenging to distinguish functionally important dynamics because proteins are inherently flexible. MMP1 is a model enzyme because both the catalytic and hemopexin domains are necessary to degrade triple-helical type-1 collagen, the highly proteolysis-resistant structural component of the extracellular matrix. We report, for the first time, measurements of MMP1 inter-domain dynamics on type-1 collagen fibrils. We have identified functionally relevant MMP1 conformations where the two domains are far apart. Mutations and ligands can allosterically modulate the dynamics that correlate with activity. The dynamics follow a two-state Poisson process that connects the picosecond timescales of MD simulations with the millisecond timescales of experiments. The two domains can functionally communicate via collagen even when the physical linker is absent.

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Section: Resultsmentioning

confidence: 99%

Section: Methods Summarymentioning

confidence: 99%

Section: Methods Summarymentioning

confidence: 99%

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Allosteric communications between domains modulate the activity of matrix metalloprotease-1

Kumar

Nash

Harms

et al. 2019

Preprint

Self Cite

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“…We cleaved off pro domains using 0.1 mg/mL trypsin to activate both active and catalytically inactive point mutant (E219Q) MMP1 as well as active MMP9 ( 26 ). We purified activated MMPs using a protease-based purification method ( 27 ). We labeled purified MMP1 with Alexa Fluor 555 (catalog no.…”

Section: Methodsmentioning

confidence: 99%

“…1 B , right panel ) on the synthetic substrate, MCA-Lys-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH 2 (catalog no. ES010; R&D Systems, Minneapolis, MN) ( 27 ). MMP9 was not labeled.…”

Section: Methodsmentioning

confidence: 99%

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

Kumar

Nash

Harms

et al. 2020

Biophysical Journal

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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.

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Solubilization and Refolding of Inclusion Body Proteins

Singhvi

Panda

2022

Methods in Molecular Biology

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Efficient protease based purification of recombinant matrix metalloprotease-1 in E. coli

Cited by 12 publications

References 64 publications

Allosteric communications between domains modulate the activity of matrix metalloprotease-1

Allosteric communications between domains modulate the activity of matrix metalloprotease-1

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

Solubilization and Refolding of Inclusion Body Proteins

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