Protein Denaturation

In some simple proteins, denaturing agents, e.g. urea, guanidinium chloride and heat, induce a two-state transition, from native to random conformation (Barone et al. 1997; Mayne and Englander 2000). However, most proteins, and in particular integral membrane proteins, can be regarded as complex systems containing independent domains that do not necessarily form cooperative structures (Arrondo and Goni 1999; Kumar and Yu 2004). Urea is a chaotropic agent that causes protein un-folding/denaturation by disrupting hydrogen bonds which hold the protein in its unique structure. Urea may also disrupt hydrophobic interactions by promoting the solubility of hydrophobic residues in aqueous solutions (Tanford 1968; Monera et al. 1994; Soloaga et al. 1998). In principle, urea cannot be used in IR studies, because

Fig. 4.7. Reversible and irreversible effects ofurea on sarcoplasmic reticulum ATPase activity. Enzyme activity was assayed on sarcoplasmic reticulum vesicles treated with varying concentrations ofurea, before (closed circles) and after (open circles) washing with urea-free buffer. Average values oftwo very similar experiments

Urea

Fig. 4.7. Reversible and irreversible effects ofurea on sarcoplasmic reticulum ATPase activity. Enzyme activity was assayed on sarcoplasmic reticulum vesicles treated with varying concentrations ofurea, before (closed circles) and after (open circles) washing with urea-free buffer. Average values oftwo very similar experiments

Urea

of its strong absorbance in the spectral region corresponding to the protein amide I band. For this reason 13C-urea, whose IR spectrum does not interfere so strongly with the protein amide I band (Reinstadler et al. 1996), has to be used. Sarcoplasmic reticulum (SR) ATPase activity was assessed in SR vesicles in the presence of urea. As seen in Fig. 4.7, 2 M urea causes a dramatic decrease in activity; higher concentrations produce further decreases. When urea is washed away after treatment, the activity is almost fully recovered for 2 and 3 M urea; however, 4 and 5 M urea appear to cause irreversible damage under our conditions, tte conformational changes associated with the activity changes can be studied by 2D-IR correlation spectroscopy using temperature as a perturbation, tte 2D-IR study whose results are summarized in Fig. 4.8 was designed primarily to understand the changes taking place during the "intermediate state" of thermal denaturation, whose temperature is 45°C (Echabe et al. 1998). For this reason the maps in Fig. 4.8 were obtained by correlating spectra between 20 and 45°C. tte control sample spectral map in the absence of urea is shown in Fig. 4.8a. Several autopeaks are seen, at 1,620, 1,632, 1,640, 1,654, 1,664, 1,675 and 1,682 cm-1, and also a number of cross-peaks, showing an important redistribution of the secondary structures, tte fact that 4 M and higher concentrations of urea lead to irreversible structural and functional changes in SR ATPase indicates a profound modification of protein conformation, including likely the transmembrane domain. However, in the presence of urea no differences are seen in this temperature region, indicating that the intermediate state (characterized by band changes at around 1,640 cm-1) is lost under these conditions, tte recovery of the correlational maps in 3M urea but not in 4M urea points to an interaction with the intramembranal domain of the ATPase. Figure 4.8b corresponds to the sample in 3M urea, ttere is only background noise, and the characteristic shift of the urea band "tail", ttus, 2D-IR correlation spectroscopy confirms the lack of an intermediate transition during SR ATPase thermal denaturation in the presence of 3 M urea, tte reversibility of the 3 M urea effect is apparent in Fig. 4.8c, corresponding to the SR vesicles treated with 3 M urea, and then washed, tte original pattern (Fig. 4.8a) is almost quantitatively recovered. Also in agreement with our previous observations, treatment with 4 M urea prevents the oligomer-monomer transition

16S0 1660

1640

16S0 1660

1640

WAVENUMBER (cm"') <600

WAVENUMBER [cm"1)

WAVENUMBER (cm"') <600

WAVENUMBER (cm1)

WAVENUMBER [cm"1)

WAVENUMBER (cm1)

1660 1060 1640 1620

WAVENUMBER (cm"')

o sr

1660 1060 1640 1620

WAVENUMBER (cm"')

1700 -1700

Was this article helpful?

0 0

Post a comment