Site-specific protein immobilization through N-terminal oxime linkages

Christman, Karen L.; Broyer, Rebecca M.; Tolstyka, Zachary P.; Maynard, Heather D.

doi:10.1039/b618002g

Cited by 80 publications

(69 citation statements)

References 49 publications

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“…MMP-1 was expressed, purified and activated, and the magnetic tweezers apparatus was calibrated as described previously in Adhikari et al 34 The N –terminus of the collagen trimer was biotinylated using PLP and biotin hydrazide as described earlier. 43–45 …”

Section: Methodsmentioning

confidence: 99%

Conformational Dynamics Accompanying the Proteolytic Degradation of Trimeric Collagen I by Collagenases

2012

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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.

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Section: Methodsmentioning

confidence: 99%

Conformational Dynamics Accompanying the Proteolytic Degradation of Trimeric Collagen I by Collagenases

2012

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“…Protein bioconjugation is used to study proteins in their biological context through fluorophore attachment (Griffin et al, 1998; O’Hare et al, 2007; Cravatt et al, 2008), improve their efficacy as therapeutic agents by conjugation to polymer chains (Zalipsky, 1995; Baker et al, 2006), and enable the construction of new types of protein-based materials (Wang et al, 2002; Christman et al, 2007; Esser-Kahn and Francis, 2008; Abedin et al, 2009). A number of reactions exist to modify the functional groups on amino acid side chains (Hermanson, 1996); however, the product mixtures that result from the indiscriminate modification of multiple sites on the protein surface are unsuitable for many applications.…”

Section: Commentarymentioning

confidence: 99%

Site‐Specific Protein Bioconjugation via a Pyridoxal 5′‐Phosphate‐Mediated N‐Terminal Transamination Reaction

Witus

Francis

2010

CP Chemical Biology

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The covalent attachment of chemical groups to proteins is a critically important tool for the study of protein function and the creation of protein‐based materials. Methods of site‐specific protein modification are necessary for the generation of well defined bioconjugates possessing a new functional group in a single position in the amino acid sequence. This article describes a pyridoxal 5′‐phosphate (PLP)–mediated transamination reaction that is specific for the N‐terminus of a protein. The reaction oxidizes the N‐terminal amine to a ketone or an aldehyde, which can form a stable oxime linkage with an alkoxyamine reagent of choice. Screening studies have identified the most reactive N‐terminal residues, facilitating the use of site‐directed mutagenesis to achieve high levels of conversion. Additionally, this reaction has been shown to be effective for a number of targets that are not easily accessed through heterologous expression, such as monoclonal antibodies. Curr. Protoc. Chem. Biol. 2:125‐134 © 2010 by John Wiley & Sons, Inc.

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“…25 We have also directly generated nanofeatures of aminooxy groups with electron beam lithography. 26 Chan and coworkers were able to immoblize ketone-decorated gold colloids onto aminooxy SAMs on a gold surface.…”

Section: Introductionmentioning

confidence: 99%