Folding, native state proteins

Through combined effects of noncovalent forces, proteins fold into secondary stmctures, and hence a tertiary stmcture that defines the native state or conformation of a protein. The native state is then that three-dimensional arrangement of the polypeptide chain and amino acid side chains that best facihtates the biological activity of a protein, at the same time providing stmctural stabiUty. Through protein engineering subde adjustments in the stmcture of the protein can be made that can dramatically alter its function or stabiUty. 

Typically, proteins fold to organize a very specific globular conformation, known as the protein s native state, which is in general reasonably stable and unique. It is this well-defined three-dimensional conformation of a polypeptide chain that determines the macroscopic properties and function of a protein. The folding mechanism and biological functionality are directly related to the polypeptide sequence a completely random amino acid sequence is unlikely to form a functional structure. In this view, polypeptide sequence... 

Scheme 4 Derivation of values in a three-state folder protein. Outline of the folding process and the folding rates are shown in the upper right inset. U denotes the unfolded state N, the folded native state and I, the folding intermedier. The respective transition states between them are denoted TSui and TSUN. The extent of relative destabilization (AG) for a particular mutant is shown above the states. Calculation of values is shown in the lower right inset. Adapted from Ref. 94.

Any polypeptide chain containing n residues could, In principle, fold Into 8 conformations. This value Is based on the fact that only eight bond angles are stereochemically allowed in the polypeptide backbone. In general, however, all molecules of any protein species adopt a single conformation, called the native state for the vast majority of proteins, the native state Is the most stably folded form of the molecule. 

Figure 13 A typical one-dimensional potential energy surface for a chemical reaction. Two stable states corresponding to the reactants A and the products B are separated by a barrier. The transition between the two states is described by a reaction coordinate q that takes on different values for the reactants and products. The rate-limiting step corresponds to reaching the conformation located at the top of the barrier qf which is the transition state. Depending on the namre of the underlying dynamical process, the rate of the reaction can be predicted accurately by one transition state theory or the Kramers formalism. In protein folding, the reactants A are associated with the unfolded state and the products B are taken to be the folded native state. The reaction coordinate typically remains unspecified. ...

MES)==10 These results suggest tliat C(MES) grows (in all likelihood) only as In N with N. Thus tlie restriction of compactness and low energy of tlie native states may impose an upper bound on tlie number of distinct protein folds. 

The key question we want to answer is what are the intrinsic sequence dependent factors tliat not only detennine tire folding rates but also tire stability of tire native state It turns out tliat many of tire global aspects of tire folding kinetics of proteins can be understood in tenns of tire equilibrium transition temperatures. In particular, we will show tliat tire key factor tliat governs tire foldability of sequences is tire single parameter... 

The biologiccJ function of a protein or peptide is often intimately dependent upon the conformation(s) that the molecule can adopt. In contrast to most synthetic polymers where the individual molecules can adopt very different conformations, a protein usually exists in a single native state. These native states are found rmder conditions typically found in Uving cells (aqueous solvents near neutred pH at 20-40°C). Proteins can be unfolded (or denatured) using high-temperature, acidic or basic pH or certain non-aqueous solvents. However, this unfolding is often reversible cind so proteins can be folded back to their native structure in the laboratory. 

Sali A, E Shakhnovich and M Karplus 1994b. PCinetics of Protein Folding. A Lattice Model Study of Requirements for Folding to the Native State, journal of Molecular Biology 235 1614-1636. 

Measuring Protein Sta.bihty, Protein stabihty is usually measured quantitatively as the difference in free energy between the folded and unfolded states of the protein. These states are most commonly measured using spectroscopic techniques, such as circular dichroic spectroscopy, fluorescence (generally tryptophan fluorescence) spectroscopy, nmr spectroscopy, and absorbance spectroscopy (10). For most monomeric proteins, the two-state model of protein folding can be invoked. This model states that under equihbrium conditions, the vast majority of the protein molecules in a solution exist in either the folded (native) or unfolded (denatured) state. Any kinetic intermediates that might exist on the pathway between folded and unfolded states do not accumulate to any significant extent under equihbrium conditions (39). In other words, under any set of solution conditions, at equihbrium the entire population of protein molecules can be accounted for by the mole fraction of denatured protein, and the mole fraction of native protein,, ie. 

For any given protein, the number of possible conformations that it could adopt is astronomical. Yet each protein folds into a unique stmcture totally deterrnined by its sequence. The basic assumption is that the protein is at a free energy minimum however, calometric studies have shown that a native protein is more stable than its unfolded state by only 20—80 kj/mol (5—20 kcal/mol) (5). This small difference can be accounted for by the favorable... 

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