Anomalous Negative Fluorescence Anisotropy in Yellow Fluorescent Protein (YFP 10C):  Quantitative Analysis of FRET in YFP Dimers

Shi, Xinghua; Basran, Jaswir; Seward, Harriet E.; Childs, W. V.; Bagshaw, Clive R.; Boxer, Steven G.

doi:10.1021/bi701575n

Cited by 39 publications

(55 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Several factors might contribute to increased homoaggregation of ErbB2 compared with ErbB1: (i) the C-terminal PDZ binding motif that interacts with intracellular proteins such as Erbin and Pick1 (29); (ii) linker regions to the mYFP moiety (26); or (iii) the mYFP moiety itself (30). Additional transgenes were constructed: ErbB2-short-mYFP, with a dipeptide linker; ACP-ErbB2, an amino terminal acyl carrier protein (ACP) sequence insertion (31); and C-terminal VPV deletion mutants of both constructs.…”

Section: Resultsmentioning

confidence: 99%

Distribution of resting and ligand-bound ErbB1 and ErbB2 receptor tyrosine kinases in living cells using number and brightness analysis

Nagy

Claus

Jovin

et al. 2010

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

164

159

View full text Add to dashboard Cite

Ligand-driven dimerizations of ErbB receptor subunits fulfill a fundamental role in their activation. We have used the number and brightness analysis technique to investigate the existence of preformed ligand-independent dimers and clusters and to characterize the initial steps in the activation of ErbB1 and ErbB2. In cells expressing 50,000-200,000 receptors, ErbB1 was monomeric in the absence of ligand stimulation, whereas in CHO cells with receptor levels >500,000 as much as 30% of ErbB1 was present as preformed dimers. EGF induced the formation of ErbB1 dimers as well as larger clusters (up to pentamers) that colocalized with clathrin-coated pits. The distribution of unstimulated ErbB2 in cells expressing 3·10 5 − 10 6 receptors was fundamentally different, in that this receptor was present in preformed homoassociated aggregates containing 5-10 molecules. These constitutive ErbB2 homoclusters colocalized with caveolae, increased in size at subphysiological temperatures, but decreased in size upon EGF stimulation. We conclude that these ErbB2 clusters are promoted primarily by membrane-mediated interactions and are dispersed upon ligand stimulation.EGFR | epidermal growth factor | ErbB proteins | receptor clusters | signal transduction E rbB proteins (ErbB1-4, HER1-4) constitute the best characterized family of receptor tyrosine kinases (1). Biochemical analysis has demonstrated that ErbB1, the prototypical member of the family (also known as the epidermal growth factor receptor, EGFR or HER1) undergoes ligand-induced homodimerization as a key step in its activation (2). More recent crystallographic studies reveal that ligand binding induces a transition from a closed conformation of ErbB1 to an extended configuration with the capacity to dimerize via intermolecular interactions mediated by domain II (3, 4). The ultrastructural data also confirm that dimerization of ErbB1 plays a fundamental role in activating the kinase domain by a mechanism resembling that of cyclin dependent kinases (5, 6). The coreceptor ErbB2 has no known ligand but upon transactivation expresses the most potent kinase activity of the ErbB family, thereby increasing the efficiency of signaling mediated by ErbB2-containing heterodimers (7). ErbB2 constitutively adopts an extended conformation potentiating the formation of heterodimers (8, 9) that can be inhibited by pertuzumab, a monoclonal antibody sterically blocking the heterodimerization arm of ErbB2 (10). Although the extracellular domain of ErbB2 has failed to form crystallographic homodimers, molecular biological and fluorescence resonance energy transfer (FRET) experiments have shown that full-length ErbB2 exists in dimers or higher-order aggregates in the plasma membrane (11,12). The implication is that the transmembrane, juxtamembrane and other intracellular domains (5, 13, 14) act in conjunction with the membrane environment (15) to mediate the dimerization and, thereby, functional states of ErbB proteins.Many investigators have sought to determine the distribution of ErbB1 in ...

show abstract

Section: Resultsmentioning

confidence: 99%

Distribution of resting and ligand-bound ErbB1 and ErbB2 receptor tyrosine kinases in living cells using number and brightness analysis

Nagy

Claus

Jovin

et al. 2010

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

164

159

View full text Add to dashboard Cite

show abstract

“…Clearly, structural interpretation of the photoselection measurements critically relies on the determination and accuracy of the vectors of both the optical transition dipole and the infrared transition dipoles belonging to the normal modes. In principle, it is possible to obtain orientational information of the optical transition dipole moment from polarized absorption measurements on crystals, which have been reported (29,30). Using P2 1 2 1 2 1 crystals, a polarization ratio of 1.6 was determined for the neutral state at 400 nm.…”

Section: Photoselection Measurements Of Ground and Excited-state Intementioning

confidence: 99%

“…Using P2 1 2 1 2 1 crystals, a polarization ratio of 1.6 was determined for the neutral state at 400 nm. This provides two solutions at 1.5°and 63.4°(assuming measurement along f100g) and two at 76.0°and À21.3°(assuming measurement along f010g) relative to a molecular reference axis that is in the chromophore plane and connects the OH and O2 chromophore atoms (30). Based on the previously reported experimental dichroism of the 1711 cm À1 transient (7), which provides a value of 6°for the angle a with the reference axis, Shi et al (30) selected the 1.5°solution as the most likely candidate form 1 : Calculation of the optical transition dipole using TD-DFT provided a dipole vector that is rotated 16°relative to the reference axis used by Shi et al (30) (Fig.…”

Section: Photoselection Measurements Of Ground and Excited-state Intementioning

confidence: 99%

Balance between Ultrafast Parallel Reactions in the Green Fluorescent Protein Has a Structural Origin

Thor

Ronayne

Towrie

et al. 2008

Biophysical Journal

View full text Add to dashboard Cite

The fluorescence photocycle of the green fluorescent protein is functionally dependent on the specific structural protein environment. A direct relationship between equilibrium protein side-chain conformation of glutamate 222 and reactivity is established, particularly the rate of ultrafast proton transfer reactions in the fluorescence photocycle. We show that parallel transformations in the photocycle have a structural origin, and we report on the vibrational properties of responsive amino acids on an ultrafast timescale. Blue excitation of GFP drives two parallel, excited-state deuteron transfer reactions with 10 ps and 75 ps time constants to the buried carboxylic acid side chain of glutamate 222 via a hydrogen-bonding network. Assignment of 1456 cm(-1) and 1441 cm(-1) modes to nu(sym) and assignment of 1564 cm(-1) and 1570 cm(-1) features to nu(asym) of E222 in the 10 ps and 75 ps components, respectively, was possible from the analysis of the transient absorption data of an E222D mutant and was consistent with photoselection measurements. In contrast to the wild-type, measurements of E222D can be described with only one difference spectrum, with the nu(sym) mode at 1435 cm(-1) and the nu(asym) mode at 1567 cm(-1), also correlating a large Deltanu(asym-sym) with slow excited-state proton transfer kinetics. Density Functional Theory calculations and published model compound and theoretical studies relate differences in Deltanu(asym-sym) to the strength and number of hydrogen-bonding interactions that are detected via equilibrium geometry and COO- stretching frequency differences of the carboxylate. The correlation of photocycle kinetics with side-chain conformation of the acceptor suggests that proton transfer from S205 to E222 controls the rate of the overall excited-state proton transfer process, which is consistent with recent theoretical predictions. Photoselection measurements show agreement for localized C=O vibrations of chromophore, Q69, and E222 with Density Functional Theory and ab initio calculations placed in the x-ray geometry and provide their vibrational response in the intermediates in the photocycle.

show abstract

“…To translate GFP transition dipole orientation to the orientation of LFA-1 in the leading edge of migrating cells we utilized the orientation of the GFP excitation dipole relative to the GFP crystal structure measured by two independent techniques 28,41 . Based on the high anisotropy of GFP crystals, and the angular dependence of their polarized excitation and emission maxima, the excitation and emission dipoles of GFP are within a few degrees of one another 28,29 .…”

Section: Resultsmentioning

confidence: 99%

Direction of actin flow dictates integrin LFA-1 orientation during leukocyte migration

Nordenfelt

Moore

Mehta

et al. 2016

Preprint

View full text Add to dashboard Cite

Integrin αβ heterodimer cell surface receptors mediate adhesive interactions that provide traction for cell migration. Here, we test whether the integrin, when engaged to an extracellular ligand and the cytoskeleton, adopts a specific orientation dictated by the direction of actin flow on the surface of migrating cells. We insert GFP into the rigid, ligand-binding head of the integrin, model with Rosetta the orientation of GFP and its transition dipole relative to the integrin head, and measure orientation with fluorescence polarization microscopy. Cytoskeleton and ligand-bound integrins orient in the same direction as retrograde actin flow with their cytoskeleton-binding β-subunits tilted by applied force. The measurements demonstrate that intracellular forces can orient cell surface integrins and support a molecular model of integrin activation by cytoskeletal force. Our results place atomic, Å-scale structures of cell surface receptors in the context of functional and cellular, μm-scale measurements.

show abstract

Anomalous Negative Fluorescence Anisotropy in Yellow Fluorescent Protein (YFP 10C): Quantitative Analysis of FRET in YFP Dimers

Cited by 39 publications

References 43 publications

Distribution of resting and ligand-bound ErbB1 and ErbB2 receptor tyrosine kinases in living cells using number and brightness analysis

Distribution of resting and ligand-bound ErbB1 and ErbB2 receptor tyrosine kinases in living cells using number and brightness analysis

Balance between Ultrafast Parallel Reactions in the Green Fluorescent Protein Has a Structural Origin

Direction of actin flow dictates integrin LFA-1 orientation during leukocyte migration

Contact Info

Product

Resources

About