Analytica Chimica Acta, accepted, 07/07/2015. This is the accepted version without proofing
corrections. DOI: 10.1016/j.aca.2015.06.011
.
Page 13 of 26
The contrast between chemical and thermal denaturation in terms of component
anisotropy (Figure 5a/b) was striking. Measureable changes in protein structure start to occur
at GuHCl concentrations of 1–1.5M and with a maximum unfolding at 4M GuHCl where a
highly random coil conformation was obtained. Anisotropy plots for each component
showed clearly that this was a single step transition between the native and the unfolded
states, because the anisotropy change was the same for each emitter. This agreed with
previously published results [53, 54], for example, initial (0M)/final (4M) anisotropy ratios
(~1.7) for both Trp components were close the mean lifetime ratio (1.9) from Flora et al.[17]
Comparing the MCR scores (Figure 3, and SI, Fig. S-12/13) shows very different behavior
and it was interesting to note that the Comp3 (RTP) contribution increased in a sigmoidal
fashion (centered at 2M GuHCl) as GuHCl concentration increased. This comparison
showed unambiguously how fundamentally different the denaturation methods are in terms of
effect on intrinsic emission and how ARMES provides another layer of information for
structural change elucidation.
3.5 Refolded structure analysis: After thermal denaturation, HSA solutions were cooled
overnight to 20 °C and aniso-TSFS data recollected. Neither anisotropy (Figure 5) nor MCR
scores (Figure 3) returned to starting values thus proving that the refolded structure after
thermal denaturation was different. For example, Comp4 anisotropy (Trp-214) at 20 °C
changed from 0.27 in the native state to r=0.31 on refolding after cooling from 70 °C. This
was clear proof that refolding of the hydrophobic pocket in subdomain IIA resulted in either
a more rigid structure where the local motion of the Trp was restricted compared to native
state, or, that the refolded state was more solvent exposed, leading to a Trp lifetime reduction
which would account for an increased anisotropy. Here, the second option was more
probable, as reports have shown that there was a decreased Trp lifetime for HSA denaturation
due to increased solvent exposure after recovery from thermal denaturation. The increased
anisotropy was ~11% which was the same as the mean lifetime decrease of 5.28 to 4.72 ns
recorded by Flora et al. [17]. This was supported by evaluation of MCR scores (Figure 3)
where Comp4 scores were always much less after refolding which indicated higher
quenching due to solvent exposure. To understand more about the refolded subdomain IIA
state we considered the RTP signal where a slightly higher anisotropy was obtained, which
again could be caused by two factors: a change in local rigidity or fluorophore lifetime.
However, MCR showed that Comp3 was always greater after refolding indicative of a more
restricted environment. This combination of fluorescence and RTP data clearly proves that
subdomain IIA, became more rigid, but solvent exposed in the refolded HSA after thermal
denaturation above 60 °C. This agreed with Mitra’s conclusions from time-resolved and
hydration studies that subdomain IIA native structure was not fully recovered upon cooling
[18].
For the Tyr based components, anisotropy recovery on refolding differed. Comp1
recovered to 0.18 (80%) indicating that subdomain IIB did not recover fully to the native
state. This agreed with observations that after cooling, the protein recovered to only ~50% αhelicity as subdomain IIB remains unfolded [18, 49]. Comp2 on the other hand recovered to
a higher anisotropy of ~ 0.24, indicating that hydrophobic subdomain IIIA refolded into a
similar, but more compact structure compared to the native state. This can be explained by
corrections. DOI: 10.1016/j.aca.2015.06.011
.
Page 13 of 26
The contrast between chemical and thermal denaturation in terms of component
anisotropy (Figure 5a/b) was striking. Measureable changes in protein structure start to occur
at GuHCl concentrations of 1–1.5M and with a maximum unfolding at 4M GuHCl where a
highly random coil conformation was obtained. Anisotropy plots for each component
showed clearly that this was a single step transition between the native and the unfolded
states, because the anisotropy change was the same for each emitter. This agreed with
previously published results [53, 54], for example, initial (0M)/final (4M) anisotropy ratios
(~1.7) for both Trp components were close the mean lifetime ratio (1.9) from Flora et al.[17]
Comparing the MCR scores (Figure 3, and SI, Fig. S-12/13) shows very different behavior
and it was interesting to note that the Comp3 (RTP) contribution increased in a sigmoidal
fashion (centered at 2M GuHCl) as GuHCl concentration increased. This comparison
showed unambiguously how fundamentally different the denaturation methods are in terms of
effect on intrinsic emission and how ARMES provides another layer of information for
structural change elucidation.
3.5 Refolded structure analysis: After thermal denaturation, HSA solutions were cooled
overnight to 20 °C and aniso-TSFS data recollected. Neither anisotropy (Figure 5) nor MCR
scores (Figure 3) returned to starting values thus proving that the refolded structure after
thermal denaturation was different. For example, Comp4 anisotropy (Trp-214) at 20 °C
changed from 0.27 in the native state to r=0.31 on refolding after cooling from 70 °C. This
was clear proof that refolding of the hydrophobic pocket in subdomain IIA resulted in either
a more rigid structure where the local motion of the Trp was restricted compared to native
state, or, that the refolded state was more solvent exposed, leading to a Trp lifetime reduction
which would account for an increased anisotropy. Here, the second option was more
probable, as reports have shown that there was a decreased Trp lifetime for HSA denaturation
due to increased solvent exposure after recovery from thermal denaturation. The increased
anisotropy was ~11% which was the same as the mean lifetime decrease of 5.28 to 4.72 ns
recorded by Flora et al. [17]. This was supported by evaluation of MCR scores (Figure 3)
where Comp4 scores were always much less after refolding which indicated higher
quenching due to solvent exposure. To understand more about the refolded subdomain IIA
state we considered the RTP signal where a slightly higher anisotropy was obtained, which
again could be caused by two factors: a change in local rigidity or fluorophore lifetime.
However, MCR showed that Comp3 was always greater after refolding indicative of a more
restricted environment. This combination of fluorescence and RTP data clearly proves that
subdomain IIA, became more rigid, but solvent exposed in the refolded HSA after thermal
denaturation above 60 °C. This agreed with Mitra’s conclusions from time-resolved and
hydration studies that subdomain IIA native structure was not fully recovered upon cooling
[18].
For the Tyr based components, anisotropy recovery on refolding differed. Comp1
recovered to 0.18 (80%) indicating that subdomain IIB did not recover fully to the native
state. This agreed with observations that after cooling, the protein recovered to only ~50% αhelicity as subdomain IIB remains unfolded [18, 49]. Comp2 on the other hand recovered to
a higher anisotropy of ~ 0.24, indicating that hydrophobic subdomain IIIA refolded into a
similar, but more compact structure compared to the native state. This can be explained by