A method for probing the topography and interactions of proteins: footprinting of myoglobin.

Zhong, Min; Lin, Laura; Kallenbach, Neville R.

doi:10.1073/pnas.92.6.2111

Cited by 34 publications

(20 citation statements)

References 38 publications

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“…The use of limited proteolysis to identify binding surfaces has been previously described for several protein interactions (37)(38)(39). In this study, the protease protection analyses provide interesting experimental data on the location of the dimer lateral interface site.…”

Section: Discussionmentioning

confidence: 78%

Initiation of Spectrin Dimerization Involves Complementary Electrostatic Interactions between Paired Triple-helical Bundles

Begg¹,

Harper²,

Morris³

et al. 2000

Journal of Biological Chemistry

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The spectrin heterodimer is formed by the antiparallel lateral association of an ␣ and a ␤ subunit, each of which comprises largely a series of homologous triplehelical motifs. Initiation of dimer assembly involves strong binding between complementary motifs near the actin-binding end of the dimer. In this study, the mechanism of lateral spectrin association at this dimer nucleation site was investigated using the analytical ultracentrifuge to analyze heterodimers formed from recombinant peptides containing two or four homologous motifs from each subunit (␣20 -21/␤1-2; ␣18 -21/␤1-4). Both the two-motif and four-motif dimer associations were weakened substantially with increasing salt concentration, indicating that electrostatic interactions are important for the dimer initiation process. Modeling of the electrostatic potential on the surface of the ␣20 and ␤2 motifs showed that the side of the motifs comprising the A and B helices is the most favorable for association, with an area of positive electrostatic potential on the AB face of the ␤2 motif opposite negative potential on the AB face of the ␣20 motif and vise versa. Protease protection analysis of the ␣20 -21/␤1-2 dimer showed that multiple trypsin and proteinase K sites in the A helices of the ␤2 and ␣21 motifs become buried upon dimer formation. Together, these data support a model where complementary long range electrostatic interactions on the AB faces of the triple-helical motifs in the dimer nucleation site initiate the correct pairing of motifs, i.e. ␣21-␤1 and ␣20-␤2. After initial docking of these complementary triple-helical motifs, this association is probably stabilized by subsequent formation of stronger hydrophobic interactions in a complex involving the A helices of both subunits and possibly most of the AB faces. The ␤ subunit A helix in particular appears to be buried in the dimer interface.Members of the spectrin family of membrane skeleton proteins are widely expressed in vertebrates as well as in lower organisms. In erythrocytes, spectrin is the major component of the membrane skeleton, a network of spectrin oligomers crosslinked with short actin filaments that is bound to the membrane and provides cell membrane stability. The most common form of spectrin on intact cell membranes is the tetramer, which is formed by the "head-to-head" association of two elongated heterodimers, each comprising an ␣ subunit and a ␤ subunit with molecular masses of 280 kDa (1) and 246 kDa (2), respectively. Both the ␣ and ␤ subunits consist largely of a series of homologous motifs, each approximately 106 residues in length (3). The tertiary structure of these motifs is a triplehelical bundle, as determined by x-ray crystallography (4) and NMR spectroscopy (5). Head-to-head tetramers form by binding of complementary partial motifs at the ends of the ␣ and ␤ subunits to form a complete triple-helical bundle (6 -8).The antiparallel lateral association of the ␣ and ␤ subunits to form a heterodimer is initiated near the actin-binding end of the molecule at a dim...

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Section: Discussionmentioning

confidence: 78%

Initiation of Spectrin Dimerization Involves Complementary Electrostatic Interactions between Paired Triple-helical Bundles

Begg¹,

Harper²,

Morris³

et al. 2000

Journal of Biological Chemistry

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“…The extension of footprinting assays to the study of protein structure was made by Heyduk and Heyduk (1) and Zhong et al (2). These studies showed that, as in the case for nucleic acids, both enzyme and chemical based cleavage reagents are suitable for determining the protected regions within a protein or in a macromolecular complex.…”

mentioning

confidence: 98%

Unfolding of Apomyoglobin Examined by Synchrotron Footprinting

Chance

2001

Biochemical and Biophysical Research Communications

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“…A protein footprinting technique has emerged for the study of protein-ligand interactions (Hanai & Wang 1994;Heyduk & Heyduk 1994;Zhong et al 1995), and has been successfully applied to the study of inter-subunit interaction in E. coli RNA polymerase (Greiner et al 1996;Heyduk et al 1996). Protein footprints of a specific complex can be obtained by the nonspecific cleavage of a complex containing the target protein, and visualization of the cleaved fragments containing one or the other terminus after fractionation on SDS-polyacrylamide gels.…”

Section: Introductionmentioning

confidence: 99%

Regions of the Escherichia coli primary sigma factor σ⁷⁰ that are involved in interaction with RNA polymerase core enzyme

Nagai

Shimamoto

1997

Genes to Cells

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Background: The j factors of bacterial RNA polymerase are required for recognition of promoters in transcription initiation. Most j factors share several regions with significant homology in their amino acid sequences (regions 1-4). Some primary j factors carry a large nonconserved segment between regions 1 and 2. The binding of an j factor to the core enzyme alters the structure and properties of the j factor, but little is known about the binding mechanism and subsequent reactions. In this report, we employed the protein footprinting method to investigate the alteration of the structure and function of Escherichia coli j 70 by binding to core enzyme and promoter DNA.

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A method for probing the topography and interactions of proteins: footprinting of myoglobin.

Cited by 34 publications

References 38 publications

Initiation of Spectrin Dimerization Involves Complementary Electrostatic Interactions between Paired Triple-helical Bundles

Initiation of Spectrin Dimerization Involves Complementary Electrostatic Interactions between Paired Triple-helical Bundles

Unfolding of Apomyoglobin Examined by Synchrotron Footprinting

Regions of the Escherichia coli primary sigma factor σ⁷⁰ that are involved in interaction with RNA polymerase core enzyme

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A method for probing the topography and interactions of proteins: footprinting of myoglobin.

Cited by 34 publications

References 38 publications

Initiation of Spectrin Dimerization Involves Complementary Electrostatic Interactions between Paired Triple-helical Bundles

Initiation of Spectrin Dimerization Involves Complementary Electrostatic Interactions between Paired Triple-helical Bundles

Unfolding of Apomyoglobin Examined by Synchrotron Footprinting

Regions of the Escherichia coli primary sigma factor σ70 that are involved in interaction with RNA polymerase core enzyme

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Product

Resources

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Regions of the Escherichia coli primary sigma factor σ⁷⁰ that are involved in interaction with RNA polymerase core enzyme