USO DE LOS PRODUCTOS ANTIALÉRGICOS

Since I was planning to use the signal of the intrinsic Trp in SH3 as a probe for studying the binding between H0 and SH3, I first determined the linear regime of the Trp in SH3 fluorescence intensity-concentration relationship to validate our research (fig. 4.4).

In order to amplify the magnitude of Trp quenching during H0/SH3 interaction for better signal to noise ratio, I introduced a Trp at position 10 in the H0 helix (through the mutation F10W). A fluorescence titration between H0 F10W and SH3 was carried out. Figure 4.5 A-D shows fluorescence emission spectra at different SH3 concentrations and a constant H0 F10W concentration. With increasing SH3 concentration, quenching of Trp emission is observed to increase. The quenching percentage of Trp emission was calculated and plotted in figure 4.5E. The data were then fitted by a Langmuir isotherm adsorption, which yielded a dissociation constant (kd) of 7.5 ± 1.9μΜ (uncertainty is from

standard error).

To exclude the possibility that quenching of Trp in SH3 was due to the introduction of PheCN or Trp in H0 helix, I next tested the influence of H0 WT helix on the signal of Trp 68

in SH3. Figure 4.5F shows the quenching percentage of Trp in SH3 with presence of H0 WT. The quenching percentages induced by H0 WT are smaller than those induced by H0 F10W, but still are mostly positive and increase with increasing SH3 concentration. The Langmuir isotherm adsorption fitting results a dissociation constant (kd) of 4.5 ±

6.0μΜ (uncertainty is from standard error). The uncertainty is relative high, which is probably due to the small quenching percentages.

D. Discussion

We investigated the autoinhibition mechanism of endophilin, by monitoring the changes of fluorescence spectra in order to reveal the interactions between H0 and SH3. The FRET experiments, which used H0 F10FCN as the donor and SH3 as the acceptor, led to an interesting phenomenon of quenching of both donor and acceptor emission. We found that this quenching happened with different donor to acceptor ratios and different donor and acceptor concentrations. Furthermore, quenching increased with increasing concentrations while the donor to acceptor ratio was held constant. Quenching of donor signal suggests that there is FRET between the H0 and SH3, which provides experimental evidences for binding between H0 and SH3.Though we did not observe sensitized emission, we believe this is due to simultaneous quenching of Trp, which is a known phenomenon of ligand binding to SH3 domains(1, 12). This quenching likely be so strong that it overcomes the sensitized emission.

Alternative to FRET experiments, we used Trp in the SH3 domain as a probe to detect the interaction between H0 and SH3. Both H0 F10W and H0 WT were proven to be able to induce quenching of the Trp in the SH3 domain. These quenching experiments further 69

prove binding between H0 and SH3 in solution. Both titrations yield similar dissociation constants, at the magnitude of ten micro molars. So far our result first reports the experimental determined binding affinity between separated H0 and SH3 of endophilin in solution. The simulation research which proposed this H0/SH3 interaction only reported a binding energy instead of a free energy of binding. The binding energy was found to be 13.0 ± 0.6 kcal/mol(9). A closer comparison might be the binding between endophilin SH3 and PP19, which is a synaptoganin derived peptide, of which the dissociation constant was reported to be 14 μM (13). This result has similar magnitude compared to our results.

In conclusion, we found experimental evidences for the interaction between H0 and SH3 domain of endophilin through three different kinds of experiments, and obtained the binding affinity between them in solution. This research should greatly contribute to understanding of auto-inhibition mechanism of endophilin in solution, and elucidate the function of SH3 domain of endophilin which is a key player in regulating other accessory proteins in CME.

Figures

Figure 4.1: H0 F10FCN/ SH3 FRET.

A. Inspection of the equilibrated H0/ SH3 complex shows that the Trp327 and Phe338 residues surround the Phe10 residue, leading to a strong hydrophobic interaction. Panel A is reproduced from(9) under permission from Publisher Elsevier Group.

B. The chemical structure of PheCN which is used to subsitute Phe10.

C. Steady-state emission spectruma of H0 F10FCN only (green hollow square), SH3 only (red sphere, rescaled according to its excitation spectrum as used in the EmEx method(14) (Briefly, the excitation spectra of the acceptor only and the acceptor with the donor were taken. The the emission spectrum of the acceptor would be rescaled according to the ratio between the excitation spectra of the acceptor without or with the donor, before compared with the emission spectrum of the acceptor and the donor)) and FRET between H0 F10FCN and SH3 (black line), respectively. H0 F10FCN was 300 μM and SH3 was 20 μM, which yielded a donor to acceptor ratio of 15:1.

D. Presenting the result in panel C in a different format: the separated spectrum of H0 F10FCN only and SH3 only in (C). were summed and gave one spectrum (red hollow sphere). Excitation wavelengths are 240nm.

Figure 4.2: Determination of the linear regime of fluorescence

intensity-concentration relationship for H0 F10FCN.

A. Emission spectra of H0 F10FCN at a series of concentrations.

B. Fluorescence intensity values at wavelength 295nm from emission spectrums in panel A at different H0 F10FCN concentrations. The vertical black line shows the boundary of linear regime. Excitation wavelengths are 240nm.

Figure 4.3: Incubation of H0 F10FCN and SH3 leads to both donor and acceptor

emission intensities decrease.

A-D. H0 F10FCN only (green hollow square), SH3 only (red sphere, rescaled according to its excitation spectrum as in the EmEx method(14)) . H0 F10FCN was mixed with SH3 domains (black line) at different solution concentrations but the same H0 F10FCN to SH3 ratio (2:1). Excitation wavelengths are 240nm.

Figure 4.4: Determine linear regime of fluorescence intensity-concentration relationship for endophilin SH3 domain.

A. Emission spectra of SH3 domains at various concentrations.

B. Fluorescence intensities at wavelength 358nm from emission spectra in panel A at different SH3 solution concentrations. The vertical black line shows the boundary of the linear regime. Excitation wavelengths are 280nm.

Figure 4.5: Titration of quenching of Trp in SH3 by H0 F10W or H0 WT.

A-D. Fluorescent emission spectra of SH3 domains at different concentrations with H0 F10W held constant at 400μM. The green line is the spectrum of incubated SH3 and H0 F10W, while the black line is the addition of separated SH3 and H0 F10W spectrums. E. Fluorescence titration experiments for SH3 and H0 F10W. Black dots are data points averaged from three independent experiments. Error bars are SEM. The data is fitted by Langmuir adsorption isotherm adsorption shown as a black line.

F. Fluorescence titration experiments for SH3 and H0 WT. Black dots are data points from three independent experiments. Error bars are SEM. The data is fitted by Langmuir adsorption isotherm shown as a black line. Excitation wavelengths are 280nm.

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