**2. Mechanism of protein folding and misfolding**

Protein folding implies nucleation condensation mechanism, wherein interaction between the residues chiefly leads to the first stage of folding process forming various transition states subsequently resulting in folding of proteins leading to formation of a stable native state [3, 11, 12]. Minimum energy levels are effective for protein folding mechanism, where the lowest energy level helps protein to achieve their stable native state [13, 14]. Initiation of the nucleation process or first phase of the nucleation condensation mechanism prevails from optimum hydrophobic and polar interactions between the formed residues. These interaction results in formation of stable globular structure of protein and serves as quality control system avoiding protein misfolding [10, 15]. Moreover, depending upon the sizes of proteins slightly different mechanisms are adopted for smaller and larger proteins. Smaller proteins undergo two-state mechanisms unlikely to larger protein, where the procedure is more complex with formation of transition states or intermediates or oligomers between the unfolded and fully folded state [16].

Various locations utilized for protein folding, such as in the cells, ribosomes are responsible for protein synthesis in a mechanism called co-translational folding, some folds in cytoplasm after complete protein synthesis, and some folds in endoplasmic reticulum (ER) or mitochondria during translocation [10]. However, some of the polypeptide regions during folding, which are otherwise buried if exposed, lead to formation of unfavorable interactions with other molecules causing protein misfolding. This explains that in protein-folding pathways transient non-native states develop to hide the regions of protein chain, which can cause aggregates on interaction with other molecules commonly the hydrophobic patches [17].

Protein misfolding mechanism and aggregation follow similar trend of seeding nucleation model [3]. This involves two processes or phases, the former phase is

*The picture represents the process that occurs, leading to protein misfolding. Picture courtsey: [21].*

called the lag phase, which is responsible for formation of oligomers causing misfolding to occur, and the later phase is called the elongation phase or exponential or polymerization phase [18]. Elaborated studies on the misfolding mechanism reveal that the initial steps are thermodynamically unfavorable and progress slowly until the minimum stable oligomeric unit is formed referred to as seeds [19]. After this step, exponential increase in the rate of formation is observed from oligomers to fibers [20]. The rate of formation can be altered by addition of preformed seeds, which minimizes the lag phase and enhances the polymerization phase. Oligomers are, thereby, considered as best seeds to propagate the misfolding process in an exponential manner (**Figure 2**) [22].
