Development of a fluorogenic sensor for activated Cdc42

Goguen, Brenda N.; Loving, Galen S.; Imperiali, Barbara

doi:10.1016/j.bmcl.2011.04.051

Cited by 20 publications

(16 citation statements)

References 21 publications

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“…Solvatochromism is a more recent development, but is becoming more widely used as new scaffolds (affinity molecules) and dyes (environmentally-sensitive fluorophores) are developed and become available (reviewed in [2]). Several groups have successfully developed solvatochromic-based biosensors using DNA aptamers [3–5], native protein receptors[6,7], peptides[8,9] or engineered binders using protein scaffolds[10–15]. However, to our knowledge, none of these groups have purposely engineered binders with affinities specified for optimal performance as a sensor, relying instead on previously-described proteins.…”

Section: Introductionmentioning

confidence: 99%

Equilibrium and dynamic design principles for binding molecules engineered for reagentless biosensors

Picciotto

Imperiali

Griffith

et al. 2014

Analytical Biochemistry

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Reagentless biosensors rely on the interaction of a binding partner and its target to generate a change in fluorescent signal using an environment sensitive fluorophore or Förster Resonance Energy Transfer. Binding affinity can exert a significant influence on both the equilibrium and the dynamic response characteristics of such a biosensor. We here develop a kinetic model for the dynamic performance of a reagentless biosensor. Using a sinusoidal signal for ligand concentration, our findings suggest that it is optimal to use a binding moiety whose equilibrium dissociation constant matches that of the average predicted input signal, while maximizing both the association rate constant and the dissociation rate constant at the necessary ratio to create the desired equilibrium constant. Although practical limitations constrain the attainment of these objectives, the derivation of these design principles provides guidance for improved reagentless biosensor performance and metrics for quality standards in the development of biosensors. These concepts are broadly relevant to reagentless biosensor modalities.

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Section: Introductionmentioning

confidence: 99%

Equilibrium and dynamic design principles for binding molecules engineered for reagentless biosensors

Picciotto

Imperiali

Griffith

et al. 2014

Analytical Biochemistry

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“…4 Previously-used dyes changed intensity but not excitation/emission maxima, so it had been necessary to attach a second, non-responsive fluorophore to the biosensor for ratio imaging. 4,25 Our new biosensor, named CRIB199 , was based on attachment of mero199 at the same position. It was assessed in vitro using the methods previously validated for the two fluorophore biosensor.…”

Section: Resultsmentioning

confidence: 99%

Ratiometric Imaging Using a Single Dye Enables Simultaneous Visualization of Rac1 and Cdc42 Activation

et al. 2016

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Biosensors that report endogenous protein activity in vivo can be based on environment-sensing fluorescent dyes. The dyes can be attached to reagents that bind selectively to a specific conformation of the targeted protein, such that binding leads to a fluorescence change. Dyes that are sufficiently bright for use at low, non-perturbing intracellular concentrations typically undergo changes in intensity rather than the shifts in excitation or emission maxima that would enable precise quantitation through ratiometric imaging. We report here mero199, an environment-sensing dye that undergoes a 33-nm solvent-dependent shift in excitation. The dye was used to generate a ratiometric biosensor of Cdc42 (CRIB199) without the need for additional fluorophores. CRIB199 was used in the same cell with a FRET sensor of Rac1 activation to simultaneously observe Cdc42 and Rac1 activity in cellular protrusions, indicating that Rac1 but not Cdc42 activity was reduced during tail retraction, and specific protrusions had reduced Cdc42 activity. A novel program (EdgeProps) used to correlate localized activation with cell edge dynamics indicated that Rac1 was specifically reduced during retraction.

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“…The presence of a fusion partner, such as SUMO, to simplify expression led to higher background fluorescence. It is noteworthy that peptide-based SuCESsFul biosensors have been found to exhibit both very low background fluorescence and high dynamic range, most likely due to the absence of strong intramolecular fluorophore interactions in the unbound state 44–46 .…”

Section: Discussionmentioning

confidence: 99%

Design Principles for SuCESsFul Biosensors: Specific Fluorophore/Analyte Binding and Minimization of Fluorophore/Scaffold Interactions

Picciotto

Dickson

Traxlmayr

et al. 2016

Journal of Molecular Biology

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Quantifying protein location and concentration is critical for understanding function in situ. SuCESsFul biosensors, in which a reporting fluorophore is conjugated to a binding scaffold, can in principle detect analytes of interest with high temporal and spatial resolution. However, their adoption has been limited due to the extensive empirical screening required for their development. We sought to establish design principles for this class of biosensor by characterizing over 400 biosensors based on various protein analytes, binding proteins and fluorophores. We found that the brightest readouts are attained when a specific binding pocket for the fluorophore is present on the analyte. Also, interaction of the fluorophore with the binding protein it is conjugated to can raise background fluorescence, considerably limiting sensor dynamic range. Exploiting these two concepts, we designed biosensors that attain a 100-fold increase in fluorescence upon binding to analyte, an order of magnitude improvement over the previously best reported SuCESsFul biosensor. These design principles should facilitate the development of improved SuCESsFul biosensors.

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Development of a fluorogenic sensor for activated Cdc42

Cited by 20 publications

References 21 publications

Equilibrium and dynamic design principles for binding molecules engineered for reagentless biosensors

Equilibrium and dynamic design principles for binding molecules engineered for reagentless biosensors

Ratiometric Imaging Using a Single Dye Enables Simultaneous Visualization of Rac1 and Cdc42 Activation

Design Principles for SuCESsFul Biosensors: Specific Fluorophore/Analyte Binding and Minimization of Fluorophore/Scaffold Interactions

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