Document Detail


Multiplexed single-molecule force proteolysis measurements using magnetic tweezers.
MedLine Citation:
PMID:  22871786     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Arjun S Adhikari; Jack Chai; Alexander R Dunn
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Video-Audio Media     Date:  2012-07-25
Journal Detail:
Title:  Journal of visualized experiments : JoVE     Volume:  -     ISSN:  1940-087X     ISO Abbreviation:  J Vis Exp     Publication Date:  2012  
Date Detail:
Created Date:  2012-08-08     Completed Date:  2012-11-02     Revised Date:  2014-08-06    
Medline Journal Info:
Nlm Unique ID:  101313252     Medline TA:  J Vis Exp     Country:  United States    
Other Details:
Languages:  eng     Pagination:  -     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Bacteriophage lambda / chemistry,  genetics
Collagen / chemistry,  metabolism
DNA, Viral / chemistry
Magnetics / instrumentation*
Matrix Metalloproteinase 1 / chemistry,  metabolism
Molecular Sequence Data
Peptide Hydrolases / chemistry*,  metabolism*
Proteolysis*
Grant Support
ID/Acronym/Agency:
1-DP2-OD007078/OD/NIH HHS
Chemical
Reg. No./Substance:
0/DNA, Viral; 9007-34-5/Collagen; EC 3.4.-/Peptide Hydrolases; EC 3.4.24.7/Matrix Metalloproteinase 1
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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