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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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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.