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A Fluorescent Probe Designed for Studying Protein A Fluorescent Probe Designed for Studying Protein Conformational ChangeConformational Change

Cohen, Bruce E. et al. (2005) Proc. Natl. Acad. Sci. USA 102, 965-970

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• Design of a fluorescent probe AminoPhenoxazone Maleimide (APM)

• Study of the probe by steady state fluorescence

• Application in 2 proteins; - Voltage gated Shaker K+ ion channel

expressed in Xenopus oocytes and - TM6 helix of β2 Αdrenergic Receptor

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• Techniques used; - TEVC Fluorometry for the Shaker K+ ion

channel- Steady State Fluorescence for purified

preparations of β2 Αdrenergic Receptor

- Life time measurements using TCSPC.

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Types of K+ Channels

• Voltage-gated• Inward Rectifying• Ca2+ sensitive• ATP-sensitive• Na+ activated• Cell volume sensitive• Type A• Receptor-coupled

Voltage-gated• 6 transmembrane domains• 4 subunits surround central pore (S5 & S6 regions of

each subunit• Selectivity filter (P region)

– Hydrophobic sequence between last 2 TMD; contains Gly-Tyr-Gly

• Voltage sensor (S4) has multiple positively charged amino acids ever 4th and 3rd positions

NH2..PVSPQDKSSNQAMSLAILRVIRLVRVFRIFKLSRHSK..COOH

• Activated by depolarization• Present in both excitable and non-excitable cells• Functions

– Regulate resting membrane potential– Control of the shape and frequency of action potentials

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346 356 359 361 365 366

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More structure• Two gating theories

– Ball and chain– Paddle

Ball and Chain Theory

When the channel is open (center), any one of the four inactivation balls can inactivate the channel (right). Inactivation for a Na+ channel is similar, but there is a single inactivaton ball.

Armstrong & Hille (1998) Neuron 20:371-380

Paddle Theory

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– where S4 lies in the lipid, at the channel periphery– and moves through the membrane– as a unit with a portion of S3

• Tested by accessibility of thiol-reactive probes– Tetramethylrhodamine maleimide (TMRM)– Methanethiosulfonate (MTS) reagents

New model of voltage sensing domain

A model of Shaker K+ gating in which the S4 segment undergoes a change in secondary structure.

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• Simple and well characterized excitation ad emission spectra.

• Significantly increase the fluorescence to describe conformational change.

• Neutral and uncharged• Having a donor and an acceptor in the same system

(opposite ends of an aromatic system Weber et al).• Ex. ANS, PRODAN, ALADAN.

Designing a fluorophore

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Synthesis of APM

O

N

NH2

MeO OMe

NHBoc

HO OH

NO

Me2N

Me2N

NH2

O O

N

Me2N

N

O

O

O

1) BBr3

2) NaOH, Boc2O

ZnCl2

O

O

O

AcOH

2) (SiMe3)2NH, ZnCl2DMF/Benzene (1:9)

1)

,

2) TFA, Thioanisole

Fig. 1 Synthesis of APM

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Fig. 2. Corrected and normalized steady-state fluorescence excitation (A) and emission (B) spectra of the ethanethiol adduct of APM in (from left to right) toluene, ethyl acetate, acetonitrile, ethanol, and water

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2 electrodes, V to monitor the membrane potential and I to inject current to hold the membrane at a desired potential. P1 and P2 are salt bridges. High salt solutions, to monitor the solution potential surrounding the oocyte.

TEV Setup

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Fig. 3. Voltage-clamp fluorometry of Xenopus oocytes expressing Shaker A359C or L361C channels

VCF Profiles

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Results

• APM can report on a known membrane protein functional rearrangement as was reported using TMRM.

• Residues labeled in the hydrophobic side gave signals with APM but no signals with TMRM. (Fig. 3C)

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β2-Adrenergic Receptors

• Prototype G-Protein Coupled Receptor (GPCR)• Exist in different conformations in addition to resting and

active states.• Important roles in agonists induced de-sensitization.• GPCR- bind agonist on cell surface to elicit cellular

response.• TM7 most important for receptor selectivity• TM6 undergoes structural changes during agonist

interaction.

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β2-Adrenergic Receptors-TM6

• Cys of interest C265; most reactive• So mutations were made as C378S and

C406S. - in β2ARC378S, C406S double mutation done;

A271C & C265A to study to APM’s ability to label in aqueous environment.

- Isoproterenol full agonist of β2AR.

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Fig. 4. Fluorescence of purified APM labeled β2AR in detergent micelles

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Drawbacks of other fluorophores

• Xanthenes:

i) Long flexible linkers; might not mirror motion of proteins alone; ii) multiple charges may perturb the local structure; iii) complicated probe-probe interactions in multimeric proteins; iv) poorly defined environmental factors give rise to ∆F during conformational change.

• Dansyl group: excitation and emission specctra overlap with cellular auto fluorescence

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Advantages of APM

• Fluorescence falls outside range of cellular auto fluorescence

• Shorter linker (3 atoms separate point of cys attachment and and nearest point of fluorophore)

• Uncharged in ground state• Larger stokes shift• Polarity dependant bathochromic shift• 231 Å3 molec. Vol. More compact than others.

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Previous studies• Oocytes expressed in Hyper (H) and De-(D)

polarizing medium.• Results of charge voltage measurement

indicatied that• S346C and A359C mutants were in resting state in H and

active in D. • R365C resting in H but only 40% active in D.• Labeling experiments show that S346 was equally labeled

in H and D, A359C significantly more labeled in D and R365C was unlabeled in H but labeled in D.

• Depolarization increased exposure of 359 and 365 to extracellular solution.

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