The Threat of Instability: Neurodegeneration Predicted by Protein Destabilization and Aggregation Propensity

Abstract

In 1961, elegant experiments by Christian Anfinsen first demonstrated that a protein, the enzyme ribonuclease A, could be reversibly denatured (unfolded) and subsequently renatured (refolded) to its functional native state [1]. This and subsequent similar findings for many other proteins gave rise to a central tenet of protein folding, that the information specifying how a protein attains its native state is encoded in its primary amino acid sequence. Thus was posed “The Protein Folding Problem,” which remains a central unsolved problem in biology: exactly how does a protein's primary sequence determine its structure and function, or its misfolding and misfunction? Until the late 1980s, protein folding was studied by relatively few scientists, and the problem was generally regarded as fundamental, but of minimal immediate, practical importance. With the advent of massive genome sequencing efforts and the concomitant recognition that many diseases are caused by single amino acid substitutions in many different proteins, the drive to understand how primary sequence governs folding and function greatly intensified. A common feature of numerous mutation-linked diseases is the deposition of misfolded protein aggregates. Extensive studies have shown that protein aggregates are often misfunctional, i.e., toxic to cells, but the nature and targets of the toxic aggregated species are not well understood and are still under intense investigation. Anfinsen's and related experiments also established that the folding of an unfolded protein is not trivial, depending strongly on solution conditions, and protein misfolding and aggregation reactions typically compete with folding reactions. Furthermore, the difference in energy between the native folded state and the unfolded state of a protein, which defines its thermodynamic stability, is typically small (20–40 kJ mol−1), corresponding to the energy of one to several noncovalent interactions (such as a hydrogen bond, or salt bridge) among the myriad of such interactions occurring in both the folded and unfolded states [2]. As a consequence, it is quite easy to significantly alter the relative proportions of protein occupying the folded versus fully or partially unfolded states, for example by substitution of a single amino acid, or by a covalent modification such as proteolysis (as is also frequently observed for proteins associated with misfolding diseases). Thus, it appears that many proteins have evolved to be only as stable as they need to be in order to remain largely folded and active, but also sufficiently unstable to be easily turned over or regulated. Some proteins are not even folded into a defined globular shape, and instead populate a fluctuating, natively unfolded state that can switch to a folded state upon binding of a suitable partner molecule. Such natively unfolded proteins have different characteristics from native globular proteins, such as a higher proportion of charged amino acids and lower proportion of hydrophobic amino acids, which decrease their tendency to aggregate [3].