CAPÍTULO 1. Generalidades de los sistemas de CCTV
1.5 La Gestión de Video
1.5.3 Monitorización usando el software de gestión de vídeo
The primary observation of the SPR data demonstrated that the ½ DPW mutant and the ¼ DPW mutant cause a substantial reduction in clathrin TD binding, compared to the WT epsin binding response, as seen in Figure 5.9.5. This reduction in response was approximately equivalent to the reduction in response of the clathrin box motifs (257, 480 and DKO) epsin 1 mutants (Figure 5.9.3). The fact that the decreased response is very similar
Mutant Names
½ DPW
¼ DPW
ΔDPW
Figure 5.9.4: Diagram illustrating the unstructured/DPW region mutants (½
DPW, ¼ DPW, ΔDPW). The ½ DPW mutant has a shorter unstructured/DPW
region by half (93 amino acids shorter) retaining all eight DPW motifs. The ¼ DPW
mutant has shortened by a quarter (60 amino acids shorter, deleting four of the
eight DPW motifs. The ΔDPW mutant has deleted the entire unstructured/DPW
region and all the eight DPW motifs.
Mutations
Shorten unstructured/DPW region (no DPW motifs deleted) by half.
Shorten unstructured/DPW region (4 DPW motifs deleted) by a quarter
Delete unstructured/DPW region (all 8 DPW motifs deleted) entirely
- WT
between the two types of epsin mutants could add further insight to the hypothesis that the unstructured/DPW region has an equivalent importance and equal binding capacity as the two clathrin box motifs. A major observation from the SPR data is how the distance of the unstructured/DPW motif and the DPW motifs separately contribute equally to epsin 1 binding to the clathrin TD. This was demonstrated when the binding response of the ¼ DPW mutant was slightly less (~ 200 response units) than ½ DPW mutant (Figure 5.9.5 (B)), demonstrating that when the distance is shortened further and when four DPW motifs have been deleted, this further decreases the ability of the epsin 1 to bind to clathrin TD. Based on these results, I propose that both the distance and the presence of DPW motifs in that unstructured/DPW region are equally important for the manner in which the epsin 1 binds to clathrin TD, until otherwise proven.
It is important to note that the overall maximum response unit of ½ DPW is 766.7 ± 244.9 RU which is the highest in all the epsin 1 mutants used with ¼ DPW at 550.0 ± 233.3 RU which is similar to the response units of 257, 480 and DKO epsin mutants as shown in Figure 5.9.5 (B). This supports the idea that the presence of the two clathrin box motifs, as in the case of ½ DPW, provides a strong binding interaction with clathrin TD, however shortening the unstructured/DPW region has clearly played a major role in reducing epsin’s ability to link multiple clathrin TDs. Shortening the unstructured/DPW region distance even further (¼ DPW mutant) demonstrates how this mutant has equal response units with mutating the clathrin box motifs individually (257 and 480) or both together (DKO).
The lowest response (174.7 ± 44.47 RU) observed in these SPR results, was the ΔDPW epsin mutant (Figure 5.9.5) where the complete unstructured/DPW region and the eight DPW motifs have been deleted. There are several possible explanations for this observation. An initial observation could be that in the ΔDPW epsin mutant the two clathrin box motifs are in a very close proximity lacking the flexibility offered from the
unstructured/DPW region. This could affect the way the two clathrin box motifs bind to clathrin TD. If these two clathrin box motifs bind to different clathrin TD sites on multiple TDs from the cooperative behaviour with the unstructured/DPW region (Morgan et al., 1999; Greene et al., 2000; Drake et al., 2000; Drake and Traub, 2001; Kalthoff et al., 2002; Dafforn and Smith, 2004), shortening the distance between the two clathrin boxes, could prevent epsin 1’s multiple TD linking interaction effect. This effect would result in a large reduction in the affinity of epsin 1 binding to clathrin TD, compared to the other mutants, as seen in the SPR data for ΔDPW mutant (Figure 5.9.5 (B)). Alternatively, if epsin 1 normally binds to multiple TD sites (CBox, ArrestinBox and W-box) on a single clathrin TD; this would not be possible with a ΔDPW epsin 1 mutant, as the shortening of the unstructured/DPW motif region would not allow long-distance interactions and the two clathrin box motifs could bind on multiple TD sites on a single ~ 5 nm sized TD. Nevertheless, taking into account the above results, I conclude by proposing that both the distance of the unstructured region, and the DPW motifs of the region are equally important for efficient interaction of epsin 1 to clathrin TD.
Figure 5.9.5: Binding of epsin 1 full-length (residues 1-575) WT and ‘unstructured/DPW’ mutants (10 μM) to GST-TD (1-363) (1 μM), to investigate epsin:clathrin interactions. (A) Binding of purified epsin 1 of WT and three
mutants (½ DPW, ¼ DPW and ΔDPW) to GST- clathrin TD immobilized on the SPR
chip via IAC method. WT binds the strongest, with an unusual binding curve, which is hypothesized not to be a 1:1 stoichiometric binding with TD. Whereas all the three mutants have a very similar binding capacity to the GST- clathrin TD with a hypothesised 1:1 stoichiometric ratio, due to the shape of the binding curve. Each experiment was carried out in a series of three repeats in randomised order. Overall, no saturation was observed in the SPR experiments, even in 1:10 molar ratio of clathrin to epsin 1, which was the recommended ratio with excess epsin 1
concentration (B) The results from (A) are plotted on a bar chart representing the
mean from the highest response value of three independent repeats (n=3) and the
standard deviation. The WT has the highest response units followed by the ½ DPW
which has a response unit of 766.7 and the ¼ DPW has a response unit of 550.0
RU. The ΔDPW has the lowest response units of 174.7 RU.