Figure 6.4: Interaction of unlabeled hsp70 and PGKA. (A) CD spectra at 20°C of 2 µM PGKA (red), 2 µM hsp70 (blue), and 1 µM:1 µM hsp70:PGKA (purple). Average of PGK* and hsp70 spectra shown in gray (B) Change in CD signal at 222 nm. Solid lines are two state thermodynamic fit. (C) Unfolding monitored by tryptophan fluorescence. The mixture of hsp70 and PGKA (purple) shows no significant deviation from the average of the individual traces (gray)
PGKA and hsp70 both undergo a structural transition as measured by tracking the change in MRE at 222 nm (Table 6.2). When hsp70 and PGKA are mixed together in a 1:1 ratio at 20 °C, no significant deviation from their average spectra is observed (Figure 6.4A). As the mixture is heated through the melting temperature, we observe a structural transition at 46°C, near the hsp70 structural transition, and no further loss of structure is observed until much higher temperatures (≈ 65°C) Both the transition temperature and amplitude of this structural change indicate that PGKA’s loss of secondary structure due to heating is mitigated by the presence of hsp70 (Figure 6.4B).
We also monitor the effect of hsp70 on PGKA’s stability using fluorescence spectroscopy. Both hsp70 and PGKA undergo a conformational transition at T <50 °C (see Table 6.2). The mixture of PGKA and hsp70 showed no deviation from the predicted average of the individual protein melting traces (see Figure 6.4C)
6.4
Discussion
Our observation of hsp70-mCherry binding to GPGK1 as it unfolds in cells is the first observation of hsp70- substrate binding in living cells. The average binding temperature of 37.5 °C is in good agreement with the measured melting temperature of the similar PGK*-FRET measured in cells and with the predicted 39.5 °C melting temperature of GPGK1 in cells [278]. We therefore see no conclusive evidence, at least in the case of GPGK1, that hsp70-mCherry accelerates unfolding from the native state. In contrast, in vitro experiments [249, 250, 301] and theoretical modeling [302] have argued that hsp70 can act to accelerate unfolding of not only aggregated assemblies but also misfolded monomers.
The binding of unfolding substrates may have significant downstream consequences in the cell aside from preventing unfavorable interactions. The observed diffusion rate of unfolded GPGK1 in U2OS cells is slower than predicted by a simple model that includes the effect of an increase in Stokes’ radius upon unfolding [278]. One possible explanation for this slow-down is that unfolded proteins are bound by other proteins in the cell, perhaps including chaperones like hsp70. Further investigation is needed to determine if chaperone binding is responsible for the slow down in diffusion and what effect, if any, this slow down has on transport-mediated processes in the cell.
Our efforts to understand the mechanism of hsp70 substrate binding and selection led us to study the GPGK-hsp70-mCherry system in vitro. In the absence of ATP we observe a significant interaction between the two proteins that is roughly correlated with substrate unfolding and this interaction is attenuated upon ATP addition (see Figure 6.3B-C), in agreement with the simple model of hsp70 substrate affinity. However, the binding temperatures observed here in the absence of ATP are 5-10°C higher than the observed protein melting temperatures (see Table 6.2), all lie above the measured hsp70-mCherry conformational transition, and the complex is not disrupted by the addition of ATP or lowering the temperature to 4 °C, where GPGK* should be able to refold (see Figure 6.3D). Finally, we observe that hsp70-mCherry and hsp70- mTFP incubated together undergo an almost identical transition at the hsp70-mCherry melting temperature in the presence or absence of ATP (see Figure 6.3A). We therefore conclude that the transition observed between GPGK1-apo hsp70-mCh is not the onset of specific hsp70-substrate binding, but rather hsp70- unfolding mediated “sticking” to unfolded protein. The temperature dependence of binding arises from the fact that the unfolding of GPGK0-GPGK2 takes place in the same temperature range as the apo-hsp70- mCherry transition and the nucelotide dependence is caused by ATP-induced stabilization of the hsp70 conformational transition.
In addition to the large transition observed both with and without ATP, we also observe an additional small transition in D/A in the presence of ATP. This was unexpected as earlier experiments have used the
addition of ATP to disrupt complex formation. This ATP-dependent transition, unlike the major change in D/A, is responsive to substrate melting temperature, at least below the 50°C conformational transition of hsp70-mCh bound to ATP. It is difficult to put this transition in the context our established understanding of hsp70-substrate binding because so little work has been done to study hsp70 binding to a protein in the process of unfolding - most studies have examined binding to short peptides or fully chemically denaturated proteins. Understanding the process of binding to a protein beginning to unfold, however, is critical to our understanding of how hsp70 acts to protect the cell - very few cellular proteins are completely unfolded at 41°C, where the heat shock response turns on.
Most studies of hsp70 substrate selection have monitored peptide binding of apo-hsp70. Our results suggest that these studies may not provide a full or accurate picture of how hsp70 interacts with as selects substrates in the cell. We find that apo hsp70-mCh does not bind GPGK conformations that are bound both in cells and, at least weakly, in the presence of ATP. The GPGK-hsp70-mCh platform developed here can be used both in vitro and in cells to develop a quantitative understanding of how hsp70 interacts with substrates and, eventually, how this interaction affects protein folding to protect the cell.
6.5
Acknowledgements
I thank Drishti Guin for assistance expressing GPGK3 and MPGK4 and for obtaining some of the data appearing in Table 6.2 and Figure 6.3C-D.