ABSTRACT
After being synthesized, proteins are subject to the complex process of folding into their compact, functional structure. In terms of chemical formality, folding is a transition from the fully extended, unfolded conformation, designated by U, into the tightly packed, native folded conformation, designated by F. This is, in its simplest mechanism, in accordance with the equation
U F Keq
(6.1)
characterized by
K
F
U
k
= =[ ] [ ]
(6.2)
where [U] and [F] are the equilibrium concentrations of the protein in the unfolded and native folded conformations, respectively, and ku and kf are the corresponding rate constants. As pointed out by Levinthal in the mid-1960s, the process of folding should not be random. A random folding is too slow and is not in accordance with the observed protein folding times. Taking a small protein consisting of 100 amino acids as an example, and assuming only three different conformations for each residue (which is a gross underestimation!), the total number of different conformations is 5 × 1047. If each conversion takes only 10−13 s, the total time to scan randomly through all possible conformations would be 1027 years-much longer than the age of the Universe. This inconsistency is often referred to as Levinthal’s paradox. The observed folding times for small proteins made up of approximately 100 amino acids are around 0.01 s. It thus appears that folding is a directed process that
6.1 Stability of the Protein Structure and Denaturation ...................................... 102 6.2 Folding Pathways .......................................................................................... 107
6.2.1 Intermediates in the Folding Pathways ............................................. 107 6.2.2 The Molten Globule .......................................................................... 109 6.2.3 Slow Steps in Protein Folding .......................................................... 112
6.3 Assisted Folding-Chaperones and Chaperonins ......................................... 116 6.4 Proteins Designed to be Marginally Stable ................................................... 120 Further Reading ..................................................................................................... 121
occurs by a multistep, progressive stabilization of various intermediate states. The resolved pathways of protein folding and the computer simulations of this process conrm this scenario. However, this does not contradict the fact that the apparently two-step processes in accordance with Equation 6.1 have frequently been observed for small proteins with fewer than 100 amino acids, but rather speaks for the possibility that the folding in these cases is too fast to detect the intermediate states. With larger proteins that fold in a timescale of seconds and even minutes, several intermediate states are readily observed. To understand the mechanism of folding, all the steps of the process should be resolved, and the kinetic constants for each transition should be determined.
