Partially unfolded denatured proteins

The ROA spectra of partially unfolded denatured hen lysozyme and bovine ribonuclease A, prepared by reducing all the disulfide bonds and keeping the sample at low pH, together with the ROA spectra of the corresponding native proteins, are displayed in Figure 5. As pointed out in Section II,B, the short time scale of the Raman scattering event means that the ROA spectrum of a disordered system is a superposition of snapshot ROA spectra from all the distinct conformations present at equilibrium. Because of the reduced ROA intensities and large... 

There is a continuing interest to improve and extend the fimctional properties range of dairy proteins to provide both health benefits and their characteristic physical behaviors under different temperature, moisture, and pH conditions so that they may be included in foods that ordinarily do not contain them. One such research area is the extrusion texturization of whey proteins, which have resulted in dairy proteins with new characteristics imparted by a controlled texturization process, depending on the application desired (Hale et al., 2002 Manoi and Rizvi, 2008 Onwulata, 2009 Onwulata et al., 1998). Protein texturization is a two-step process that involves, first, the unfolding of the globular structure (denaturation) and, second, the alignments of the partially unfolded structures in the direction of mass flow in the extruder. The surface characteristics are imparted at the extruder die as the molten mass exits (Onwulata et al., 2003a). 

The application of ROA to studies of unfolded and partially folded proteins has been especially fruitful. As well as providing new information on the structure of disordered polypeptide and protein sequences, ROA has provided new insight into the complexity of order in denatured proteins and the structure and behavior of proteins involved in misfold-ing diseases. All the ROA data shown in this chapter have been measured in our Glasgow laboratory because, at the time of writing, ROA data on typical large biomolecules had not been published by any other group. We hope that this review will encourage more widespread use of ROA in protein science. 

The conformational plasticity supported by mobile regions within native proteins, partially denatured protein states such as molten globules, and natively unfolded proteins underlies many of the conformational (protein misfolding) diseases (Carrell and Lomas, 1997 Dobson et al., 2001). Many of these diseases involve amyloid fibril formation, as in amyloidosis from mutant human lysozymes, neurodegenerative diseases such as Parkinson s and Alzheimer s due to the hbrillogenic propensities of a -synuclein and tau, and the prion encephalopathies such as scrapie, BSE, and new variant Creutzfeldt-Jacob disease (CJD) where amyloid fibril formation is triggered by exposure to the amyloid form of the prion protein. In addition, aggregation of serine protease inhibitors such as a j-antitrypsin is responsible for diseases such as emphysema and cirrhosis. 

The denaturation of proteins generally involves at least partial unfolding, with the loss of secondary and tertiary structure. In the present context, we are interested in the end point of this process — proteins that are unfolded to the maximal extent by various agents heat, cold, acid, urea, Gdm-HCl.1 Three major questions concerning unfolded proteins are of interest in the present chapter. Do different unfolding agents... 

Protein structures have evolved to function in particular cellular environments. Conditions different from those in the cell can result in protein structural changes, large and small. A loss of three-dimensional structure sufficient to cause loss of function is called denaturation. The denatured state does not necessarily equate with complete unfolding of the protein and randomization of conformation. Under most conditions, denatured proteins exist in a set of partially folded states that are poorly understood. 

Many proteins can be made to clump into fibrous amyloid deposits like those seen in Alzheimer s disease, Creutzfeldt-Jakob disease (the human counterpart of mad cow disease), and other serious ailments. To help prove this point, a natural enzyme to convert to amyloid fibrils—insoluble protein aggregates with a /3-pleated sheet structure—simply by maintaining protein for some time in the unfolded state. Until now, scientists have generally believed that only specific proteins such as amyloid /3-protein and prions are capable of being converted into amyloid fibrils.11 A variety of spectroscopic techniques have been used to confirm the gradual development of amyloid fibrils and to verify the fibrils predominant /3-pleated sheet structure. In the partially unfolded intermediates that form under denaturing conditions, hydrophobic amino acid residues and polypeptide backbone normally buried inside fully folded structures become exposed. Further work is needed to confirm and advance these findings. 

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. 

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... 

CD spectroscopy has also provided valuable insight into the chemical stability and chemical denaturation of proteins. A recent study by Rumfeldt etal. examines the guanidinium-chloride induced denaturation of mutant copper-zinc superoxide dismutases (SODs). These mutant forms of the Cu, Zn-SOD enzyme are associated with toxic protein aggregation responsible for the pathology of amyotrophic lateral sclerosis. In this study, CD spectroscopy was used in conjunction with tryptophan fluorescence, enzyme activity, and sedimentation experiments to study the mechanism by which the mutated enzyme undergoes chemical denaturation. The authors found that the mutations in the enzyme structure increased the susceptibihty of the enzyme to form partially unfolded destabilized monomers, rather than the stable metaUated monomer intermediate or native metallated dimer. 

Figure 3.57. Components of a Partially Denatured Protein Solution. In a half-unfolded protein solution, half the molecules are fully folded and half are fully unfolded.

---