Preparation of Isotopically Labeled Recombinant Fragments of Fibronectin for Functional and Structural Study by Heteronuclear Nuclear Magnetic Resonance Spectroscopy - Methods in Molecular Biology: Extracellular Matrix Protocols - page 56

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Preparation of Isotopically Labeled Recombinant
Fragments of Fibronectin for Functional
and Structural Study by Heteronuclear Nuclear
Magnetic Resonance Spectroscopy
Jeremy R. Bright, Andrew R. Pickford, Jennifer R. Potts,
and Iain D. Campbell
1. Introduction
The collagens, proteoglycans, and glycoproteins of the extracellular matrix
(ECM) are a diverse collection of macromolecules, heterogeneous in size,
structure, and function. However, their primary structures have revealed that
their polypeptide components are frequently comprised of a series of sequence
repeats or “modules” (1) . This mosaic nature of ECM macromolecules permits
their properties to be analyzed by a “dissection” strategy (2) , where protein
fragments generated by proteolysis, chemical synthesis, or recombinant
expression are employed in structural and functional investigations.
Fibronectin, an ubiquitous ECM glycoprotein, plays a major role in funda-
mental biological processes such as cell adhesion and migration, maintenance
of normal cell morphology, cytoskeletal organization, and cell differentiation
(3 , 4) . Fibronectin is constructed from three types of independently folding pro-
tein module (F1, F2, and F3) (5) and is found as a fibrillar network in the ECM
where it interacts with other ECM components and provides anchorage sites
for cell surface integrin receptors. It remains the focus of much structural
investigation by both X-ray crystallography and nuclear magnetic resonance
(NMR) spectroscopy (6-12) .
NMR spectroscopy is a powerful technique that can be used to determine
the three-dimensional structure of small proteins and protein modules in solu-
tion and can also provide information on protein folding pathways, enzyme