Kinetics of Protein-Protein Interactions at the Surface of an Optical Biosensor

Edwards, P. R.; Gill, Anita; Pollard-Knight, Denise; Hoare, Mike; Buckle, P.E.; Lowe, Peter A.; Leatherbarrow, Robin J.

doi:10.1006/abio.1995.1522

Cited by 172 publications

(151 citation statements)

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“…Analysis of Data-In keeping with the procedure adopted in most biosensor studies with the IAsys instrument (35), the approximation has been made that the extent of complex formation between added oligoribonucleotide (O) and the immobilized hnRNP A2 is sufficiently small to allow substitution of the total RNA concentration (C O ) tot for its free counterpart, C O . The reliability of this approximation was checked by means of the following expression (Equation 1),…”

Section: Methodsmentioning

confidence: 99%

Binding of an RNA Trafficking Response Element to Heterogeneous Nuclear Ribonucleoproteins A1 and A2

Shan

Moran-Jones

Munro

et al. 2000

Journal of Biological Chemistry

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Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 binds a 21-nucleotide myelin basic protein mRNA response element, the A2RE, and A2RE-like sequences in other localized mRNAs, and is a trans-acting factor in oligodendrocyte cytoplasmic RNA trafficking. Recombinant human hnRNPs A1 and A2 were used in a biosensor to explore interactions with A2RE and the cognate oligodeoxyribonucleotide. Both proteins have a single site that bound oligonucleotides with markedly different sequences but did not bind in the presence of heparin. Both also possess a second, specific site that bound only A2RE and was unaffected by heparin. hnRNP A2 bound A2RE in the latter site with a K d near 50 nM, whereas the K d for hnRNP A1 was above 10 M. UV cross-linking assays led to a similar conclusion. Mutant A2RE sequences, that in earlier qualitative studies appeared not to bind hnRNP A2 or support RNA trafficking in oligodendrocytes, had dissociation constants above 5 M for this protein. The two concatenated RNA recognition motifs (RRMs), but not the individual RRMs, mimicked the binding behavior of hnRNP A2. These data highlight the specificity of the interaction of A2RE with these hnRNPs and suggest that the sequence-specific A2RE-binding site on hnRNP A2 is formed by both RRMs acting in cis.The family of more than 20 heterogeneous nuclear ribonucleoproteins (hnRNPs) 1 appears to play diverse roles in the post-transcriptional processing of hnRNA and subsequent packaging, transport, and translation of mRNA (1-4). hnRNPs A1, A2, B1, B2, C1, and C2 are the major components of 40S "core particles," studied primarily in HeLa cells, which are thought to package nascent ssRNA in the nucleus in a histonelike fashion (5-10). The hnRNP A/B proteins have a modular structure with two N-terminal RNA recognition motifs (RRMs) followed by a glycine-rich region ( Fig. 1) (11). The three-dimensional structures of the tandem RRMs of hnRNP A1, which are assumed to be the principal mediators of the RNA-protein interactions, have been determined (12, 13). Although the three-dimensional structures of other hnRNPs of the A/B family have not been determined, their RRMs share sufficient sequence identity with those of hnRNP A1 to suggest that they share the same tertiary fold: a four-stranded antiparallel ␤-sheet flanked by two helices. This module adopts a comparable fold in the RRMs of more distantly related proteins, such as U1A (14 -17).Although a number of the hnRNPs have a general role in intranuclear packaging of RNA, some also manifest sequencespecific binding (1, 4, 18 -22). In addition to being a major component of core particles (23), hnRNP A2 binds a telomeric sequence (24, 25, 43) and a small RNA segment known to be necessary and sufficient for transport of the message encoding myelin basic protein in the cytoplasm of oligodendrocytes, the A2RE (26 -28). The binding of hnRNP A2 to A2RE has been shown, by mutational and antisense approaches, to be essential for cytoplasmic RNA trafficking in oligodendrocytes (29).Although hnRNP A2 has been shown to ...

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Section: Methodsmentioning

confidence: 99%

Binding of an RNA Trafficking Response Element to Heterogeneous Nuclear Ribonucleoproteins A1 and A2

Shan

Moran-Jones

Munro

et al. 2000

Journal of Biological Chemistry

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“…Edwards et al (46) have found that at low concentrations of analyte a single exponential model is sufficient to describe the binding data, whereas at high concentrations a double exponential model is required. This has been attributed to the dextran surface of the biosensor chip.…”

Section: ␣-Tropomyosin's Exon 6 Affects Actin Affinitymentioning

confidence: 99%

The Sequence of the Alternatively Spliced Sixth Exon of α-Tropomyosin Is Critical for Cooperative Actin Binding but Not for Interaction with Troponin

Hammell

Hitchcock‐DeGregori

1997

Journal of Biological Chemistry

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Tropomyosins, a family of highly conserved coiled-coil actin binding proteins, can differ as a consequence of alternative expression of several exons (Lees-Miller, J., and Helfman, D. (1991) BioEssays 13, 429 -437). Exon 6, which encodes residues 189 -213 in long, 284-residue tropomyosins, has two alternative forms, exon 6a or 6b, both highly conserved throughout evolution. In ␣-tropomyosin, exon 6a or 6b is not specific to any one of the nine isoforms. Exon 6b encodes part of a putative Ca 2؉ -sensitive troponin binding site in striated muscle tropomyosins, suggesting that the exon 6-encoded region may be specialized for certain tropomyosin functions.A series of recombinant, unacetylated tropomyosin exon 6 deletion and substitution mutants and chimeras was expressed in Escherichia coli to determine the requirements of exon 6 for tropomyosin function. Functional properties of the tropomyosins were defined by actin affinity measured by cosedimentation, troponin T affinity using a newly developed biosensor assay, and regulation of the actomyosin MgATPase. The region of tropomyosin encoded by exon 6 affects actin affinity but not thin filament assembly, troponin T binding, or regulation with troponin. The tropomyosins with exon 6a or 6b function normally whether a striated muscle exon 9a or smooth/non-muscle exon 9d is present. However, the effect of deleting 21 amino acids encoded by exon 6 or replacing it with a GCN4 leucine zipper sequence depends on the COOH-terminal sequence. Tropomyosins (TM)1 are a family of highly conserved coiledcoil actin binding proteins present in most eukaryotic cells. At least 15 different isoforms arise through the use of alternative promoters and alternative RNA splicing of the transcripts of a small number of genes (three to four in vertebrates; reviewed in Ref. 1). These isoforms are expressed in developmentally and tissue-specific patterns and differ in actin affinity.A function common to all TMs is cooperative binding to F-actin (2). Tropomyosin molecules are aligned head-to-tail in the grooves of the helical actin filament (3, 4). The role of TM is best understood in striated muscle where it regulates Ca 2ϩ -dependent muscle contraction with Tn (reviewed in Refs. 5-7). Structural studies have shown that Tn, found only in striated muscles, extends along at least the COOH-terminal third of the TM molecule (8 -13). Troponin I, TnC, and the COOH terminus of TnT are positioned near Cys-190 of TM. The elongated NH 2 terminus of TnT extends beyond the COOH terminus of one TM to the NH 2 -terminal 10 -30 residues of the next TM along the actin filament (13,14).Troponin greatly increases the affinity of TM for actin in the presence of Ca 2ϩ , with a further increase upon removal of Ca 2ϩ (15). Based on binding studies with TM and Tn peptides, Mak and Smillie (16) proposed that the ␣-TM-Tn interaction in the region of Cys-190 is weakened in the presence of Ca 2ϩ (Ca 2ϩ -sensitive), whereas that at the COOH terminus and the overlap region is strong in the presence and absence of Ca 2ϩ (...

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“…Biacore is one example of a class of optical biosensors which can be used to determine the kinetic binding parameters of molecular interactions, such as association rate constant (kassoc), dissociation rate constant (kdissoc) and binding constant (Ka) [29,30]. Biacore measurements are usually performed with one partner immobilized (in the case of triplex formation, duplex) on a porous hydrogel to which the second component (in the case of triplex formation, TFO) then binds [29].…”

Section: Kinetic Analyses By Biacorementioning

confidence: 99%

“…The resultant plot was fitted to a straight line by a linear least-squares method. The association rate constant (kassoc) was determined from the slope of the fitted line [29,30]. The off-rate constant (koff) was obtained from the analysis of each dissociation curve ( Figure 6a).…”

Section: Kinetic Analyses Of Pyrimidine Motif Triplex Formation Involmentioning

confidence: 99%

“…The off-rate constant (koff) was obtained from the analysis of each dissociation curve ( Figure 6a). As koff is usually independent of the concentration of the injected solution, the dissociation rate constant (kdissoc) was determined by averaging koff for several concentrations [29,30]. Ka was calculated from the equation, Ka = kassoc/kdissoc.…”

Section: Kinetic Analyses Of Pyrimidine Motif Triplex Formation Involmentioning

confidence: 99%

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Chemical Modification of Oligonucleotides: A Novel Approach Towards Gene Targeting

Torigoe¹,

Imanishi²

2012

Mutagenesis

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Kinetics of Protein-Protein Interactions at the Surface of an Optical Biosensor

Cited by 172 publications

References 0 publications

Binding of an RNA Trafficking Response Element to Heterogeneous Nuclear Ribonucleoproteins A1 and A2

Binding of an RNA Trafficking Response Element to Heterogeneous Nuclear Ribonucleoproteins A1 and A2

The Sequence of the Alternatively Spliced Sixth Exon of α-Tropomyosin Is Critical for Cooperative Actin Binding but Not for Interaction with Troponin

Chemical Modification of Oligonucleotides: A Novel Approach Towards Gene Targeting

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