Protein Conformation and Diagnostic Tests: The Prion Protein Brian J. Bennion and Valerie Daggett * Background: Many clinical diagnostic tests depend on the accurate detection and quantification of proteins and peptides and their functions. Alterations of protein structure, and the resulting consequences on dynamics, can affect the outcome of laboratory tests. These changes can be a result of mutations, other in vivo factors, or even the experimental conditions of the diagnostic test. Approach: The relationship between protein structure and dynamics and experimentally observable properties used in diagnostic assays are discussed in light of transmissible spongiform encephalopathies, or prion diseases. Content: This review describes current efforts and pos- sible future directions of prion diagnostic development. Recent advances in therapeutic development are also addressed. Summary: The intent of the review is to highlight the role of protein dynamics and conformational change in protein-based diagnostics and treatments for prion disease. © 2002 American Association for Clinical Chemistry Protein Conformation and Dynamics in Protein-based Diagnostic Assays Most proteins assume a distinct three-dimensional struc- ture in vivo and in vitro, and this native structure is necessary for function. A protein’s structure is deter- mined by its amino acid sequence and the surrounding environment. There are various levels of structure: pri- mary structure refers to the amino acid sequence; second- ary structure refers to the fold of the peptide, i.e., -heli- ces, -strands, and turns; and tertiary structure describes the three-dimensional structure and is determined by the packing of the secondary structural elements and the amino acid side chains. As a protein or peptide unfolds, interactions are disrupted and both secondary and ter- tiary structure can be lost. In addition, the folded and unfolded states of proteins are in equilibrium. Even under conditions that favor the folded state, a protein is not locked into a single conformation. The amino acids are free to interact and move according to the forces placed on them by neighboring atoms and the solvent, producing many conformations that may differ only very slightly (conformers). Protein motion occurs on a wide spectrum with respect to time. Bond vibration and rotations occur on a femto- second timescale (10 -15 s), whereas amino acid side chains move on a picosecond to nanosecond timescale (10 -12 –10 -9 s). Small segments of the peptide backbone can reposition themselves in a matter of nanoseconds to microseconds (10 -9 –10 -6 s). The arrangement of surface amino acids can be affected by such dynamic behavior. Such changes in the surface of a protein can alter potential binding sites for other molecules. Complete folding of a protein can occur in microseconds to milliseconds or hours, depending on the sequence and solvent environ- ment. Furthermore, proteins and peptides can adopt different, folded structures under different conditions (1–3 ). Even slight changes in binding epitopes attributable to either localized or more dramatic conformational changes of a protein can have profound effects on anti- body-based diagnostic tests. This issue is particularly pertinent and potentially problematic in the case of the development of diagnostic tests for transmissible spongi- form encephalopathies, or prion diseases. Conformational Change and Disease: Transmissible Spongiform Encephalopathies There is currently much interest in transmissible spongi- form encephalopathies (TSEs) 1 because of outbreaks of Department of Medicinal Chemistry, Box 357610, University of Washing- ton, Seattle, WA 98195-7610. *Author for correspondence. E-mail daggett@u.washington.edu. Received May 9, 2002; accepted September 19, 2002. 1 Nonstandard abbreviations: TSE, transmissible spongiform encephalop- athy; BSE, bovine spongiform encephalopathy; CJD, Creutzfeldt–Jakob dis- ease; CWD, chronic wasting disease; PrP C , cellular prion protein; PrP Sc , scrapie prion protein; GPI, glycosylphosphatidylinositol; FTIR, Fourier transform infrared; NMR, nuclear magnetic resonance; FDA, Food and Drug Adminis- tration; EDRF, erythroid differentiation-related factor; and LTBR, lymphotoxin -receptor. Clinical Chemistry 48:12 2105–2114 (2002) Review 2105