Elevated temperature unfolding

The most widely used approach by far, however, is elevated temperature unfolding simulations, which are discussed in more detail below. 

Folded proteins can be caused to spontaneously unfold upon being exposed to chaotropic agents, such as urea or guanidine hydrochloride (Gdn), or to elevated temperature (thermal denaturation). As solution conditions are changed by addition of denaturant, the mole fraction of denatured protein increases from a minimum of zero to a maximum of 1.0 in a characteristic unfolding isotherm (Fig. 7a). From a plot such as Figure 7a one can determine the concentration of denaturant, or the temperature in the case of thermal denaturation, required to achieve half maximal unfolding, ie, where... 

Some are inducible by conditions that cause unfolding of newly synthesized proteins (eg, elevated temperature and various chemicals)... 

An explanation for these observances in the cases of ethanol and PEG may arise from hydrophobic interactions (i.e., methylene groups, methyl) with the unfolded state of the protein at elevated temperatures. This idea is supported by studies of the interaction of several alkylureas (methyl-, ACVdimethyl-, ethyl-, and butylureas) with the thermal unfolding of ribonuclease A, where it was shown... 

Several studies since then have supported this suggestion, and now it is widely accepted that conformational change/structural perturbation is a prerequisite for amyloid formation. Structural perturbation involves destabilization of the native state, thus forming nonnative states or partially unfolded intermediates (kinetic or thermodynamic intermediates), which are prone to aggregation. Mild to harsh conditions such as low pH, exposure to elevated temperatures, exposure to hydrophobic surfaces and partial denaturation using urea and guanidinium chloride are used to achieve nonnative states. Stabilizers of intermediate states such as trimethylamine N-oxide (TMAO) are also used for amyloidogenesis. However, natively unfolded proteins, such as a-synuclein, tau protein and yeast prion, require some structural stabilization for the formation of partially folded intermediates that are competent for fibril formation. Conditions for partial structural consolidation include low pH, presence of sodium dodecyl sulfate (SDS), temperature or chemical chaperones. 

The description above bears some resemblance to the reversible folding/unfolding of proteins. A speculative conclusion from this analogy would be that an increase in temperature should be followed by an increased exposure of hydrophobic side-chains. Hypothetically, an increase in bitter taste, relative to a standard quinine solution, should be observed at elevated temperatures whether this is found in practice or not remains to be investigated. 

From electric birefringence measurements it was concluded that the proteins are ordered head-to-tail within the fibril, in a helical configuration (Rogers et al. 2005). The fact that one needs a minimal temperature in order to induce fibrillisation is directly related to the fact that at a certain elevated temperature the protein will partially unfold. Since we have also observed the formation of fibrils at 4°C, after having applied this (partial) denaturation step, the elevated temperature is not essential during assembly. However, at the lower temperature, the assembly was found to be much slower, indicating that temperature affects the kinetics of the assembly process. The relation between the fibrillar type of assembly and the partially unfolded state also has been found for other proteins (e.g., ovalbumin, hen egg white... 

The unfolding process is a function of temperature. Many proteins have optimal stability in the temperature range 10-30°C. Loss of structure is expected both at low temperatures (cold denaturation) and at elevated temperatures. 

The change in the rate constants with temperature for the reactions of ribonuclease (RNase) with hydrated electrons and OH radicals was measured. The RNase molecule unfolds reversibly at elevated temperatures exposing sites particularly reactive towards hydrated electrons. The theoretical treatment leads to an estimate of the encounter frequencies for differently shaped macromolecules with small radiolytically produced solvent radicals. The derived encounter frequencies are compared with experimentally determined rate constants. Values as high as 1013 M"1 secr1 are understandable. 

At elevated temperatures the RNase molecule undergoes a transition from a folded to an unfolded state (5). This can be shown by optical and viscosity measurements. The transition temperature depends on pH and ionic strength. Above this temperature the tertiary structure is destroyed but the covalent disulfide bonds remain intact. The unfolding is reversible since on lowering the temperature the native conformation is regained. 

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