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. 2011 Apr 13;19(4):566-76.
doi: 10.1016/j.str.2011.01.011.

Beyond the random coil: stochastic conformational switching in intrinsically disordered proteins

Ucheor B Choi  1 , James J McCann, Keith R Weninger, Mark E Bowen
Affiliations

Beyond the random coil: stochastic conformational switching in intrinsically disordered proteins

Ucheor B Choi et al. Structure. .

Abstract

Intrinsically disordered proteins (IDPs) participate in critical cellular functions that exploit the flexibility and rapid conformational fluctuations of their native state. Limited information about the native state of IDPs can be gained by the averaging over many heterogeneous molecules that is unavoidable in ensemble approaches. We used single molecule fluorescence to characterize native state conformational dynamics in five synaptic proteins confirmed to be disordered by other techniques. For three of the proteins, SNAP-25, synaptobrevin and complexin, their conformational dynamics could be described with a simple semiflexible polymer model. Surprisingly, two proteins, neuroligin and the NMDAR-2B glutamate receptor, were observed to stochastically switch among distinct conformational states despite the fact that they appeared intrinsically disordered by other measures. The hop-like intramolecular diffusion found in these proteins is suggested to define a class of functionality previously unrecognized for IDPs.

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Figures

Figure 1
Figure 1. Charge Hydropathy Ratio for IDPs used in this Study
Mean hydropathy per residue was calculated using normalized Kyte Doolittle scale with a five residue scanning window. This is plotted against the net charge per residue. The C/H ratio has been suggested to dictate the degree of compaction in IDPs (Mao et al., 2010; Muller-Spath et al., 2010). The dotted line represents an empirically-determined charge/hydropathy relationship that distinguishes most globular and disordered proteins (Uversky, 2002). SNAP-25 (solid circle), synaptobrevin (open circle), neuroligin (open square), NMDAR-2B (solid triangle), and complexin (solid square). The C/H ratio was calculated for the polypeptide sequence neglecting the contribution of the dyes, which would introduce systematic shifts for all samples (Muller-Spath et al., 2010).
Figure 2
Figure 2. smFRET measurements of SNAP-25
(a) Schematic of Rrms measurement in IDPs. Stars represent dye positions. Distance between the two labeling sites when fully extended is termed the contour length (L) while the root mean squared (rms) distance on a diffusing IDP is denoted Rrms. (b) A double-labeled molecule is encapsulated inside a biotinylated liposome which is then immobilized by tethering to a layer of biotinylated BSA and streptavidin deposited on a quartz microscope slide. (c) Representative smFRET traces of S25(177). (d) Histograms of smFRET for S25(119) in open circles and S25(177) in solid circles. Solid line is the fit to a Gaussian distribution. See also Figures S1 and S2.
Figure 3
Figure 3. smFRET measurements of synaptobrevin
(a) Histograms of smFRET for the SB cytosolic domain in liposomes. Solid circles, SB(67). Open circles, SB(44). Solid line, Gaussian fit. (b) Schematic of doubly labeled full length SB reconstituted in PC or PC with 15% PS bilayer. (c) and (d) Comparison of smFRET for full length SB and the SB cytosolic domain for SB(44) and SB(67), respectively. Open circles, SB cytosolic domain free in liposomes. Solid squares, full length SB in 100% PC bilayer. Open triangles, full length SB in 85% PC 15% PS bilayer. See also Figures S1 and S3.
Figure 4
Figure 4. Circular dichroism measurements
Circular dichroism (CD) spectra of neuroligin (NL), open circles. SNAP-25(S25), open triangles. Glutamate receptor CTD2 (N2B), solid circles.
Figure 5
Figure 5. smFRET measurements of the neuroligin cytoplasmic domain
(a) Histograms of smFRET for NL. NL(105), solid circles. NL(59), open circles. (b) Representative smFRET as a function of time for NL(105). Fluorescence intensity, top. Calculated FRET, bottom. (c) Percent of NL(105) and NL(59) molecules displaying transitions in the 10–100 seconds before photobleaching. (d) Lifetimes of the stable FRET states between transitions in NL. NL(105), solid circles. NL(59), open circles. See also Figures S1 and S5.
Figure 6
Figure 6. Effect of PSD-95 interactions on smFRET transitions in neuroligin
(a) Histogram of smFRET measurements of NL(92) alone, open circles, and NL(92) co-encapsulated with 20-fold molar excess of unlabeled PSD-95, solid squares. (b) Schematic of a double-labeled NL co-encapsulated with unlabeled PSD-95 inside a surface tethered liposome. (c) Histogram of the of dwell times of distinct FRET states for NL(92) alone, open circles, and NL(92) with PSD-95, solid circles. Solid lines are single exponential fits with rate constants of 1.19 s−1 for NL(92) alone and 1.05 s−1 for NL(92) co-encapsulated with PSD-95. (d) Percent of NL(92) molecules observed to have at least one FRET state transition before dye bleaching during a 100 sec. observation window in the absence and presence of PSD-95. See also Figures S1, S4 and S6.
Figure 7
Figure 7. smFRET measurements of glutamate receptor cytoplasmic domain
(a) Histograms of smFRET in N2B. Three different contour lengths between donor and acceptor dye molecules were measured. Open circles, N2B(15). Solid circles, N2B(121). Open triangles, N2B(172). (b) Representative smFRET as a function of time for N2B(121). Fluorescence intensity, top. Calculated FRET, bottom. (c) Lifetimes of the stable FRET states between transitions in N2B(121). (d) Plots of dwell times in distinct FRET states as a function of FRET efficiency for single molecules of N2B(121). (e) The FRET state before a transition (y-axis) plotted against the FRET state after that transition (x-axis) for N2B(121).
Figure 8
Figure 8. Effect of binding to SNARE complex reconstituted in a lipid bilayer on the smFRET histograms of complexin
(a) Histogram of smFRET for CX encapsulated in liposomes. CX(69), solid circles. CX(32), open circles. (b) Schematic of doubly labeled CX binding to unlabeled SNARE complexes reconstituted into PC bilayer. (c) and (d) Comparison of smFRET in CX when encapsulated in liposomes (open circles) or bound to the SNARE complex on lipid bilayers (solid squares). (c) CX(32). (d) CX(69)
Figure 9
Figure 9. Schematic representation of energy landscapes that could generate steady and hop-like intramolecular diffusion in the native state of IDPs
Conformational space is represented on the x-axis while free energy is represented on the y-axis. (a) SNAP-25 where all conformations are of roughly equal energy with small barrier separating conformations. (b) Neuroligin where all conformations are still equal but the height of the barrier is increased, which would slow transitions. (c) Hypothetical restricted chain where physiological conditions change the energy landscape to favor a subset of possible conformations.
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