Thermal unfolding intermediates

The Thermal Unfolding Intermediate Is Partially Unfolded at its N-terminus. The mobility of the intermediate in the SDS gel as well as its sedimentation behavior in a sucrose gradient indicated that 7 is still trimeric (40). The slight increase in the gel electrophoretic mobility presumably resulted from increased binding of SDS molecules due to local unfolding. The partial unfolding also makes this species sediment slightly slower than the native trimer. 

The purified tailspikes can bind to phage capsids in vitro and make the resulting phage particles infectious (42). The thermal unfolding intermediate was unable to bind to the phage capsid (40). The specific binding activity to phage... 

Table I. Comparison between the Folding Intermediate (Protrimer) and the Thermal Unfolding Intermediate (/)...

B.-L. and King, J., unpublished results). Tailspike also unfolds in cold SDS solution (4°C) and converts to an intermediate species similar to the thermal unfolding intermediate with a half time of 10 days. However the mechanism of unfolding induced by urea and GdnHCl are still unclear and currently under investigation. 

During the forward folding pathway the newly synthesized chain forms a partially folded single chain intermediate (5435). This folds further to a species with sufficient structure for chain recognition. Three of these intermediates associate into a protrimer in which the chains are associated but not fully folded (36). This species then folds further to the thermostable detergent and protease resistant native trimer. There are no known covalent modifications in the process. A similar process occurs in refolding urea denatured chains in vitro (37). Thermal Unfolding Pathway of Tailspike... 

Analysis of the products of the thermal unfolding reaction revealed that after the appearance of the unfolding intermediate, recovery of the chains decreased due to aggregation. This aggregation probably resulted from the partially unfolded monomeric chains formed from the dissociation of the mtermediates. The aggregation of the partially denatured chains rendered the kinetic analysis difficult. 

Many proteins are capable of populating partially folded states under specific solution conditions and, occasionally, coexistence of the folded and an unfolded state can be observed by NMR. Ding et al have reported amide residual dipolar couplings (RDCs), nitrogen transverse relaxation rates for varying pH values and different temperatures on the destabilized mutant of the B1 domain of protein G (GBl). The RDCs for the low pH, thermally unfolded state of GBl are very small and do not indicate the presence of any native-like structure. Their data provided clear evidence for intermediate conformations and multi-state equilibrium unfolding for this GBl variant ... 

As previously reported, lysozyme under thermal unfolding [13-15] exhibits intermediate structures. (These can also be induced by pressure and chemical changes [74].) Its unfolding process is thus a three-state model A U. Reversible... 

The Streptomyces subtilisin inhibitor is closely related to the Kazal inhibitor family (Laskowski and Kato, 1980). The subunits of this dimeric inhibitor dissociate during thermal unfolding (Takahashi and Sturtevant, 1981), when dilute solutions (2--5 mg/ml) are heated slowly (l C/min) in a Privalov (1980) type of DSC instrument. The Td for Strepton yces subtilisin inhibitor is 17 C above that for STI at this heating rate (Table II), suggesting that it is intermediate in stability among the inhibitors tested. 

Thermodynamics for the sequence with the native state shown in Fig. 4 with the contact interaction potentials By taken from Table III of Ref. 54 reveals that it folds cooperatively in an apparent two-state manner. This is also reflected in the thermal distribution of the overlap function values /i(x) at the folding transition temperature 7> (Fig. 4). A nearly bimodal distribution of h(y) with the peaks at X 0.2 (NBA) and x 0-6 (unfolded state) is observed. There is also nonneghgible contribution from the intermediate values of % representing partially folded structures. Experiments that probe in more detail the thermal unfolding of proteins are beginning to reveal the possible importance of these... 

It appears from different research reports (Tanford, 1970 Pace, 1975) that, for many proteins, reversible transitions from the unfolded to the folded state have characteristics of a two-state process, without detectable intermediates at equilibrium, even when the existence of intermediates is supported by other experimental arguments (kinetics for example, see Chapters 7, 8 and 9). Urea and GuHCl denaturation is generally closer to a a two-state mechanism than pH or thermal unfolding. 

Experimentally, temperature is commonly used to bring about systematic structural changes in the RNA. To illustrate, consider the thermal equilibrium between a population of folded and unfolded RNA molecules, F U (for now ignore the existence of intermediates), governed by the equilibrium constant Keq = c j/cF. It can be shown that the change in concentration of the folded population is related to a temperature change by (Bernasconi, 1976)... 

Although less frequently discussed, heat processes often influence the textures and chemistries of the intermediate and end products, and thermal treatments are not without consequences on milk proteins that are denatured. Denaturation of proteins occurs under precise conditions of pH, temperature and ionic strength leading to their unfolding. Denaturation is significantly slower when proteins are near their isoelectric point. Only (3-lactoglobulin is irreversibly denatured at pH 7 and 70°C a-lactalbumin is denatured at pH 6.7 and 65°C. Aggregation of these proteins, besides hydrophobic... 

Figure 11.15c shows the resolved concentration profiles and spectra coming from the row-wise appended matrix containing data from the three techniques mentioned previously. The need for one additional intermediate conformation has been proven to be necessary to explain the protein folding process of a-apolactalbumin. Additionally, the thermal range of occurrence and the evolution of this intermediate can now be known. The resolved spectrum obtained for the a-apolactalbumin intermediate shows that it has an ordered secondary structure similar to the native folded protein and an unordered tertiary structure similar to the unfolded protein at high temperatures. These spectral features match the spectral description attributed to the molten globular state and provide additional evidence to confirm the presence of this species as a real intermediate conformation. 

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