Unfolded protein

In the development of new products, optimization of the fermentation medium for titer only often ignores the consequences of the medium properties on subsequent downstream processing steps such as filtration and chromatography. It is imperative, therefore, that there be effective communication and understanding between workers on the upstream and downstream phases of the produc t development if rational trade-offs are to be made to ensure overall optimahty of the process. One example is to make the conscious decision, in collaboration with those responsible for the downstream operations, whether to produce a protein in an unfolded form or in its native folded form the purification of the aggregated unfolded proteins is simpler than that of the native protein, but the refolding process itself to obtain the product in its final form may lack scalabihty. 

We will discuss three different approaches to engineer a more thermostable protein than wild-type T4 lysozyme, namely (1) reducing the difference in entropy between folded and unfolded protein, which in practice means reducing the number of conformations in the unfolded state, (2) stabilizing tbe a helices, and (3) increasing the number of bydropbobic interactions in tbe interior core. 

Denaturation is accompanied by changes in both physical and biological properties. Solubility is drastically decreased, as occurs when egg white is cooked and the albumins unfold and coagulate. Most enzymes also lose all catalytic activity when denatured, since a precisely defined tertiary structure is required for their action. Although most denaturation is irreversible, some cases are known where spontaneous renaturation of an unfolded protein to its stable tertiary structure occurs. Renaturation is accompanied by a full recovery of biological activity. 

Chaperones. Figure 2 The multiple roles of BiP in the biogenesis of the secretory proteins. BiP, immunoglobulin heavy chain binding protein ER, endoplasmic reticulum ERAD, ER-associated degradation ERj, resident ER protein with J-domain Sec61, core subunit of the protein translocase UPR, unfolded protein response that involves several signal transduction pathways that are activated in order to increase the biosynthetic capacity and decrease the biosynthetic burden of the ER... 

Unfolded protein response, when mis-folded proteins accumulate in the ER, signal transduction pathways are activated that increase the biosynthetic capacity and decrease the biosynthetic burden of the ER. 

Bochkareva, E,S., Lisson, N.M., Girshovich, A.S. (1988). Transient association of newly synthesized unfolded proteins with the heat shock GroEL protein. Nature 336, 254—257. 

Intracellular products can be present either as folded, soluble proteins or as dense masses of unfolded protein (inclusion bodies). For these products, it is first necessary to concentrate the cell suspension before effecting release of the product. Filtration can result in a suspension of cells that can be of any desired concentration up to 15 to 17 percent and that can be diafiltered into the desired buffer system. In contrast, the cell slurry that results from centrifugation will be that of... 

Absorption of proteins in the 230-300 nm range is determined by the aromatic side chains of tyrosine (Xmax = 274 am), tryptophan (Xmax = 280 nm), and phenylalanine (Xmax = 257 nm). Because the difference in the absorption spectra of native and unfolded protein molecules is generally small, difference spectra can... 

Another interesting application area of PHMD simulations is to investigate electrostatic interactions in the unfolded states of proteins. A traditional view that unfolded proteins adopt random conformational states that are devoid of electrostatic and hydrophobic interactions, are recently challenged by experimental data [20, 69], REX-CPHMD folding simulations of the 35 residue C-terminal subdomain of the villin headpiece domain revealed a significant deviation from the standard pKa values for several titratable residues. Additional simulations, in which a charged group is neutralized confirmed the existence of specific electrostatic interactions in the unfolded states (JK and CLB, manuscript in preparation). 

Toward a Taxonomy of the Denatured State Small Angle Scattering Studies of Unfolded Proteins by Millett et al. assesses denatured states induced by heat, cold, and solvent for evidence of residual structure, while Insights into the Structure and Dynamics of Unfolded Proteins from NMR by Dyson and Wright describes their extensive investigations of residual structure in the unfolded state. 

Three theory papers are also included. Determinants of the Polyproline II Helix from Modeling Studies by Creamer and Campbell reexamines and extends an earlier hypothesis about Pn and its determinants. Hydration Theory for Molecular Biophysics by Paulaitis and Pratt discusses the crucial role of water in both folded and unfolded proteins. Unfolded State of Peptides by Daura et al. focuses on the unfolded state of peptides studied primarily by molecular dynamics. 

UNFOLDED PROTEINS STUDIED BY RAMAN OPTICAL ACTIVITY... 

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