221
8 Two-Dimensional Mid-Infrared Correlation Spectroscopy in Protein Research
cA5 measured in the absence or presence of Ca
2+
ion, two populations of α-helical
structures have been identified, i.e., the canonical α-helices (1,650 cm
−1
) and solvated α-helices with those α-helices in helix-helix interactions (1,634 cm
−1
). the
results strongly support results obtained from circular dichroism and differential
scanning calorimetry; these results reveal more compact tertiary structures, which
have higher thermal stability, after calcium binding.
the formation of amyloid fibrils from the native state of the activation domain of
human procarboxypeptidase A2 (AdA2h) and three mutants with different aggregation propensities was studied by Cerdà-Costa et al. with temperature as the perturbing agent [81]. A sample at pd 3 was heated from 20 to 90 °C in 10 min to trigger the aggregation process. Next, IR spectra were measured every min for 30 min
while the sample temperature was maintained at 90 °C. unlike the studies described
above, in this case, temperature was not the direct perturbation. the temperature
change initiated the time-evolving process of fibril formation, which was the direct
perturbation. Inspection of the two distinct autopeaks at 1,616 cm
−1
and centered
around 1,647 cm
−1
, which are associated with the amyloid fibril and partially unfolded α-helices, respectively, allowed for a detailed analysis of the conversion of
the secondary structure of AdA2h from the initial monomer to the amyloid fibril.
the sequential unfolding events of horse, cow, and tuna ferricytochrome c (fcyt
c) were determined, and whether the thermal unfolding processes for the structurally homologous proteins proceeded by the same steps was examined [53]. 2dCoS
has helped to find subtle but significant differences in the order of the thermal unfolding events for the three proteins, despite their high sequence identity (> 97 %).
In cow fcyt c, the unfolding of all of the helical components occurred cooperatively,
but in horse and tuna fcyt c, the helical components behaved as subdomains that
unfolded separately.
A few papers were published by Yan et al. in which the temperature-perturbed
infrared spectra of myoglobin [61], hemoglobin [64, 69], and bovine pancreatic
ribonuclease A [74] were analyzed by 2dCoS to describe the heat-induced unfolding and aggregation processes for the three systems. the phenomenon of protein
aggregation attracts many researchers because this process is strictly correlated with
protein fibrillation [127]. Although a two-state model for thermal denaturation of
myoglobin arose from traditional 1d analysis, subtle noncooperative conformational changes identified at low temperatures by 2dCoS forced the postulate that
myoglobin aggregates under conditions when native structures dominate [61]. the
appearance of an aggregated β-structure before the unfolding of α-helices was surprising at that time. In contrast, the sequence of events from the 2d analysis of the
temperature-dependent spectra of hemoglobin clearly indicated a two-stage thermal
transition [64]. the aggregation process is preceded by the exposure of the helical
structures to the solvent; next, from 44–54 °C aggregated forms evolved from the
exposed elements. the process is completed at 70 °C by the unfolding of structures
deeply buried in the hydrophobic interior. 2dCoS analysis of the thermal denaturation of RNase A in acidic (pd 6.0) and basic (pd 8.0) conditions [74] has revealed
that the aggregation was closely associated with the oligomerization of RNase A
and that both processes proceeded at high temperatures. the sequence of events for
8 Two-Dimensional Mid-Infrared Correlation Spectroscopy in Protein Research
cA5 measured in the absence or presence of Ca
2+
ion, two populations of α-helical
structures have been identified, i.e., the canonical α-helices (1,650 cm
−1
) and solvated α-helices with those α-helices in helix-helix interactions (1,634 cm
−1
). the
results strongly support results obtained from circular dichroism and differential
scanning calorimetry; these results reveal more compact tertiary structures, which
have higher thermal stability, after calcium binding.
the formation of amyloid fibrils from the native state of the activation domain of
human procarboxypeptidase A2 (AdA2h) and three mutants with different aggregation propensities was studied by Cerdà-Costa et al. with temperature as the perturbing agent [81]. A sample at pd 3 was heated from 20 to 90 °C in 10 min to trigger the aggregation process. Next, IR spectra were measured every min for 30 min
while the sample temperature was maintained at 90 °C. unlike the studies described
above, in this case, temperature was not the direct perturbation. the temperature
change initiated the time-evolving process of fibril formation, which was the direct
perturbation. Inspection of the two distinct autopeaks at 1,616 cm
−1
and centered
around 1,647 cm
−1
, which are associated with the amyloid fibril and partially unfolded α-helices, respectively, allowed for a detailed analysis of the conversion of
the secondary structure of AdA2h from the initial monomer to the amyloid fibril.
the sequential unfolding events of horse, cow, and tuna ferricytochrome c (fcyt
c) were determined, and whether the thermal unfolding processes for the structurally homologous proteins proceeded by the same steps was examined [53]. 2dCoS
has helped to find subtle but significant differences in the order of the thermal unfolding events for the three proteins, despite their high sequence identity (> 97 %).
In cow fcyt c, the unfolding of all of the helical components occurred cooperatively,
but in horse and tuna fcyt c, the helical components behaved as subdomains that
unfolded separately.
A few papers were published by Yan et al. in which the temperature-perturbed
infrared spectra of myoglobin [61], hemoglobin [64, 69], and bovine pancreatic
ribonuclease A [74] were analyzed by 2dCoS to describe the heat-induced unfolding and aggregation processes for the three systems. the phenomenon of protein
aggregation attracts many researchers because this process is strictly correlated with
protein fibrillation [127]. Although a two-state model for thermal denaturation of
myoglobin arose from traditional 1d analysis, subtle noncooperative conformational changes identified at low temperatures by 2dCoS forced the postulate that
myoglobin aggregates under conditions when native structures dominate [61]. the
appearance of an aggregated β-structure before the unfolding of α-helices was surprising at that time. In contrast, the sequence of events from the 2d analysis of the
temperature-dependent spectra of hemoglobin clearly indicated a two-stage thermal
transition [64]. the aggregation process is preceded by the exposure of the helical
structures to the solvent; next, from 44–54 °C aggregated forms evolved from the
exposed elements. the process is completed at 70 °C by the unfolding of structures
deeply buried in the hydrophobic interior. 2dCoS analysis of the thermal denaturation of RNase A in acidic (pd 6.0) and basic (pd 8.0) conditions [74] has revealed
that the aggregation was closely associated with the oligomerization of RNase A
and that both processes proceeded at high temperatures. the sequence of events for