Disulfide bonds unfolded protein state

Of the 20 reduced or disulfide bond-free proteins for which the Rg of the chemically or thermally unfolded state has ben reported, 17 fall on a single curve (Fig. 4). Fitting the Rg of these 17 proteins produces a strong, statistically significant correlation (r 2 = 0.96) and an exponent, v = 0.61 d= 0.03, startlingly close to the expected value for an excluded-volume random coil chain. 

Disulfide bonds stabilize proteins by constraining the unfolded conformation and thereby raising the free energy of the unfolded state. In addition to the covalent peptide backbone and occasional disulfide bonds, a number of weak and subtle forces cooperate to maintain the 3-D structure of an enzyme. These forces also play a key role in the interaction of an enzyme with its substrates. These weak forces include H-bonds, electrostatic forces between charged groups, and hydro-phobic or van der Waals forces among nonpolar groups (see Section I,B,4,g in this chapter). 

Disulfides. The introduction of disulfide bonds can have various effects on protein stability. In T4 lyso2yme, for example, the incorporation of some disulfides increases thermal stability others reduce stability (47—49). Stabili2ation is thought to result from reduction of the conformational entropy of the unfolded state, whereas in most cases the cause of destabili2ation is the introduction of dihedral angle stress. In natural proteins, placement of a disulfide bond at most positions within the polypeptide chain would result in unacceptable constraint of the a-carbon chain. 

Example The cleavage of disulfide bonds by reduction with 1,4-dithiothreitol causes the unfolding of the protein. This exposes additional basic sites to protonation, and therefore results in higher average charge states in the corresponding ESI spectrum (Fig. 11.14). [88]... 

Despite many years of experimental and theoretical studies devoted to it, the protein folding problem remains essentially unsolved because there are too many conformations that can occur in both the unfolded and the folded structure to be searched. The problem of protein folding is further compounded by transient disulfide bonds, solvent, and environmental effects in general that may play an important role in stabilizing particular folded states such as, e.g., the a- or the 3i0-helix. 

Many proteins that are completely unfolded In 8 M urea and p-mercaptoethanol (which reduces disulfide bonds) spontaneously renature (refold) Into their native states when the denaturing reagents are removed by dialysis. Because no cofactors... 

Recently there have also appeared studies on the thermodynamics of the Ca2+-loaded partially unfolded state (272), the importance of the disulfide bonds (273), and the functional role of calcium-binding residues (274). Furthermore, aLAC has been a model for protein folding because it forms a molten globule state during folding (275, 276), which will not be discussed here. 

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