TOPICAL PAPER Protein Misfolding and Aggregation Regina M. Murphy* ,† and Brent S. Kendrick ‡ Department of Chemical and Biological Engineering, University of Wisconsin, Madison, Wisconsin 53706, and Amgen, Inc., Thousand Oaks, California 91320 Interest in the problem of protein misfolding and aggregation has exploded in recent years for two reasons: (1) the sharp rise in the number and volume of therapeutic proteins produced commercially and (2) the recognition of the central role of protein aggregates in degenerative diseases. The systematic study of protein aggregation presents major challenges to both the experimentalist and the theoretician. Much of the work retains an empirical flavor due to the experimental complexities; the sensitivity of protein aggregation to the slightest change in protein amino acid composition, solvent properties, or protein concentration; and the lack of robust theoretical models of misfolding and aggregation. Novel experimental and computational approaches are being developed, and we anticipate substantial progress will be made in the near future. Several presentations describing the latest advances in protein misfolding and aggregation were given at the American Chemical Society meeting (BIOT division) held in September, 2006 in San Francisco. Introduction After assembly on the ribosome, newly synthesized polypep- tide chains rapidly proceed through secondary, tertiary, and (sometimes) quaternary structure formation, to be released as stable, natively folded, functional proteins. The side chains of the incorporated amino acids display a diversity of chemical moieties: acids, bases, alcohols, amides, aromatics, alkyls (both branched and straight-chain), and sulfur-containing groups. Covalent modifications such as glycosylation, phosphorylation, deamidation, and oxidation further increase the chemical diversity. Multivalent hydrogen bonding in the polyamide backbone stabilizes the secondary structural elements and gives rise to the familiar R-helix and -sheet. Tertiary and quaternary structure arise primarily from the forces among these side chains. The burial of hydrophobic side chains is of particular impor- tance, but Coulombic interactions, including salt bridges between acids and bases, disulfide bond formation, or dipole-ion interactions such as occur between tyrosine and charged side chains, also contribute to protein folding and structural stability. Despite the enormous number of possible configurations of a polypeptide chain, folding to the native state is typically rapid and robust. A finely tuned balance of hydrophobic and Cou- lombic forces among the different parts of the polypeptide backbone, the side chains, and the solvent is required to maintain correct folding. Sometimes, however, subtle changes in the balance of forces lead to misfolded and aggregated proteins. Surprisingly small changes may produce remarkably different outcomes. For example, a point mutation as seemingly innocu- ous as glycine-to-alanine renders superoxide dismutase aggrega- tion-prone (1); as another example, bovine serum albumin is destabilized against thermal unfolding in the presence of high concentrations of chaotropic salts but stabilized at low chaotrope concentrations (2). Problems with aggregation arise routinely in the manufacture of protein products (3). Recombinant proteins produced in E. coli are typically synthesized in inclusion bodies. Although the inclusion bodies afford high productivity and rapid isolation of the crude protein, the protein is misfolded and insoluble. Solubilization and unfolding is achieved through the addition of denaturants such as urea or guanidinium chloride and reductants such as dithiothreitol. The protein is refolded by removal of the denaturant via dilution, diafiltration, or dialysis, often under optimized redox conditions. Misfolding and ag- gregation often accompany this refolding step, especially if it is carried out at high protein concentrations. Manufacturers are increasingly switching to using eukaryotic cells for protein production, because the cellular machinery facilitates processing of the polypeptide to its correct folded structure and incorpora- tion of naturally occurring carbohydrate moieties. Despite this, unwanted aggregation frequently occurs during purification, formulation, and/or vial-filling. A further challenge is encoun- tered with the need to maintain proteins in their soluble and aggregation-free state for months to years. Concerns about misfolded and aggregated proteins in therapeutic preparations range from unpredictable changes in protein activity to the very real danger of a life-threatening immunological response in the patient (4). During the past decade or two the role of protein misfolding and aggregation in at least 40 diseases has been recognized (5). The case can be made for a causative role of protein aggregation in numerous diseases including Alzheimer’s disease (-amyloid * To whom correspondence should be addressed. Ph: (608) 262-1587. Fax: (608) 262-5434. E-mail: regina@engr.wisc.edu. † University of Wisconsin, Madison. ‡ Amgen, Inc. 548 Biotechnol. Prog. 2007, 23, 548-552 10.1021/bp060374h CCC: $37.00 © 2007 American Chemical Society and American Institute of Chemical Engineers Published on Web 04/11/2007