Equilibrium Analysis of High Affinity Interactions Using BIACORE

Myszka, David G.; Jonsen, Matthew D.; Graves, Barbara J.

doi:10.1006/abio.1998.2937

Cited by 112 publications

(75 citation statements)

References 11 publications

(16 reference statements)

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“…This result is similar to the 23-fold auto-inhibition observed previously on the SC1 site (Table 1) (27). Also, as expected from previous studies, Ets-1 affinity for the Mo-MLV site was 10-fold lower than that for the high-affinity SC1 site (24,43,47). The detection of autoinhibition on the Mo-MLV enhancer establishes that this phenomenon is not dependent on the sequence or affinity of the binding site.…”

Section: Ets-1 Binding To the Mo-mlv Enhancer Is Repressed Bysupporting

confidence: 90%

Auto-Inhibition of Ets-1 Is Counteracted by DNA Binding Cooperativity with Core-Binding Factor α2

Goetz

Speck

et al. 2000

Molecular and Cellular Biology

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119

127

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Auto-inhibition is a common transcriptional control mechanism that is well characterized in the regulatory transcription factor Ets-1. Autoinhibition of Ets-1 DNA binding works through an inhibitory module that exists in two conformations. DNA binding requires a change in the inhibitory module from the packed to disrupted conformation. This structural switch provides a mechanism to tightly regulate Ets-1 DNA binding. We report that the Ets-1 partner protein core-binding factor ␣2 (CBF␣2; also known as AML1 or PEBP2) stimulates Ets-1 DNA binding and counteracts auto-inhibition. Support for this conclusion came from three observations. First, the level of cooperative DNA binding (10-fold) was similar to the level of repression by auto-inhibition (10-to 20-fold). Next, a region necessary for cooperative DNA binding mapped to the inhibitory module. Third, an Ets-1 mutant with a constitutively disrupted inhibitory module did not bind DNA cooperatively with CBF␣2. Furthermore, two additional lines of evidence indicated that CBF␣2 affects the structural switch by direct interactions with Ets-1. First, the retention of cooperative DNA binding on nicked duplexes eliminated a potential role of through-DNA effects. Second, cooperative DNA binding was observed on composite sites with altered spacing or reversed orientation. We suggest that only protein interactions can accommodate this observed flexibility. These findings provide a mechanism by which CBF relieves the auto-inhibition of Ets-1 and illustrates one strategy for the synergistic activity of regulatory transcription factors.Auto-inhibition modulates a variety of transcription factor activities (21). In this regulatory mechanism, inhibitory sequences act in cis to repress such functions as DNA binding, transcriptional activation, nuclear localization, and ligand interaction. The picture emerging from the study of several transcription factors suggests that protein partnerships can counteract auto-inhibition. For example, serum response factor counteracts the auto-inhibition of Elk-1 DNA binding (36). The DNA binding of Pip, which contains an auto-inhibitory domain, requires interactions with PU.1 (9), and Pbx activates the DNA binding of its partner protein Hoxb (10, 11). Similarly, DNA binding by the yeast repressor a1 requires formation of a ternary complex with ␣2 (63). Examples within basal transcriptional machinery include the auto-inhibition of the subunit of Escherichia coli RNA polymerase. Interaction with core RNA polymerase counters this inhibitory effect (13). Also, the inhibitory function of the amino terminus of TATAbinding protein is abrogated by interactions with SNAPc (40). In each of these DNA binding scenarios, auto-inhibition generates a tighter control switch from the off to on state. To decipher the molecular basis of the interplay between autoinhibition and protein partnerships, we focus on Ets-1, for which auto-inhibition is well characterized at the mechanistic and structural levels (21).Auto-inhibition, together with protein partnerships, c...

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Section: Ets-1 Binding To the Mo-mlv Enhancer Is Repressed Bysupporting

confidence: 90%

Auto-Inhibition of Ets-1 Is Counteracted by DNA Binding Cooperativity with Core-Binding Factor α2

Goetz

Speck

et al. 2000

Molecular and Cellular Biology

Self Cite

119

127

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“…2B). Thus, continuous microfluidic washing appears to be significantly more effective at removing nonspecific phage, presumably by eliminating rebinding events (33)(34)(35)(36). This finding is further supported by the work of Yuan et al which used flow-based phage selection in an surface plasmon resonance (SPR) instrument (37).…”

Section: Resultsmentioning

confidence: 75%

Selection of phage-displayed peptides on live adherent cells in microfluidic channels

Wang

Teesalu

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Affinity reagents that bind to specific molecular targets are an essential tool for both diagnostics and targeted therapeutics. There is a particular need for advanced technologies for the generation of reagents that specifically target cell-surface markers, because transmembrane proteins are notoriously difficult to express in recombinant form. We have previously shown that microfluidics offers many advantages for generating affinity reagents against purified protein targets, and we have now significantly extended this approach to achieve successful in vitro selection of T7 phagedisplayed peptides that recognize markers expressed on live, adherent cells within a microfluidic channel. As a model, we have targeted neuropilin-1 (NRP-1), a membrane-bound receptor expressed at the surface of human prostate carcinoma cells that plays central roles in angiogenesis, cell migration, and invasion. We show that, compared to conventional biopanning methods, microfluidic selection enables more efficient discovery of peptides with higher affinity and specificity by providing controllable and reproducible means for applying stringent selection conditions against minimal amounts of target cells without loss. Using our microfluidic system, we isolate peptide sequences with superior binding affinity and specificity relative to the well known NRP-1-binding RPARPAR peptide. As such microfluidic systems can be used with a wide range of biocombinatorial libraries and tissue types, we believe that our method represents an effective approach toward efficient biomarker discovery from patient samples.

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“…We used surface plasmon resonance to measure the kinetics of the interaction between HuD and AU-rich target RNAs. This powerful approach, which has only begun to be used to study RNA-protein interactions and has never been applied to the study of Hu proteins, can visualize complex formation in real time and can provide unique insights into the dynamics of association and dissociation (40). HuD was chosen for our studies because the function of its RNA-binding domains has been best characterized (14,35).…”

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confidence: 99%

HuD RNA Recognition Motifs Play Distinct Roles in the Formation of a Stable Complex with AU-Rich RNA

Park

Myszka

et al. 2000

Molecular and Cellular Biology

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. Hu proteins can bind to AU-rich elements in the 3 untranslated regions of unstable mRNAs, causing the stabilization of certain transcripts. We have studied the interaction between HuD and prototype mRNA instability elements of the sequence UU(AUUU) n AUU using equilibrium methods and real-time kinetics (surface plasmon resonance using a BIACORE). We show that a single molecule of HuD requires at least three AUUU repeats to bind tightly to the RNA. Deletion of RRM1 reduced the K d by 2 orders of magnitude and caused a decrease in the association rate and a strong increase in the dissociation rate of the RNA-protein complex, as expected when a critical RNA-binding domain is removed. In contrast, deletion of either RRM2 or -3, which only moderately reduced the affinity, caused marked increases in the association and dissociation rates. The slower binding and stabilization of the complex observed in the presence of all three RRMs suggest that a change in the tertiary structure occurs during binding. The individual RRMs bind poorly to the RNA (RRM1 binds with micromolar affinity, while the affinities of RRM2 and -3 are in the millimolar range). However, the combination of RRM1 and either RRM2 or RRM3 in the context of the protein allows binding with a nanomolar affinity. Thus, the three RRMs appear to cooperate not only to increase the affinity of the interaction but also to stabilize the formed complex. Kinetic effects, similar to those described here, could play a role in RNA binding by many multi-RRM proteins and may influence the competition between proteins for RNA-binding sites and the ability of RNA-bound proteins to be transported intracellularly.

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Equilibrium Analysis of High Affinity Interactions Using BIACORE

Cited by 112 publications

References 11 publications

Auto-Inhibition of Ets-1 Is Counteracted by DNA Binding Cooperativity with Core-Binding Factor α2

Auto-Inhibition of Ets-1 Is Counteracted by DNA Binding Cooperativity with Core-Binding Factor α2

Selection of phage-displayed peptides on live adherent cells in microfluidic channels

HuD RNA Recognition Motifs Play Distinct Roles in the Formation of a Stable Complex with AU-Rich RNA

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