Uncorrected Proof 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 JOURNAL OF MOLECULAR RECOGNITION J. Mol. Recognit. 2004; 17: 1–9 DOI:10.1002/jmr.699 The effect of macromolecular crowding on protein aggregation and amyloid fibril formation Larissa A. Munishkina, Elisa M. Cooper, Vladimir N. Uversky and Anthony L. Fink* Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064, USA Macromolecular crowding is expected to have several significant effects on protein aggregation; the major effects will be those due to excluded volume and increased viscosity. In this report we summarize data demonstrating that macromolecular crowding may lead to a dramatic acceleration in the rate of protein aggregation and formation of amyloid fibrils, using the protein -synuclein. The aggregation of -synuclein has been implicated as a critical factor in development of Parkinson’s disease. Various types of polymers, from neutral polyethylene glycols and polysaccharides (Ficolls, dextrans) to inert proteins, are shown to accelerate -synuclein fibrillation. The stimulation of fibrillation increases with increasing length of polymer, as well as increasing polymer concentration. At lower polymer concentrations (typically up to  100 mg/ml) the major effect is ascribed to excluded volume, whereas at higher polymer concentrations evidence of opposing viscosity effects become apparent. Pesticides and metals, which are linked to increased risk of Parkinson’s disease by epidemiological studies, are shown to accelerate -synuclein fibrillation under conditions of molecular crowding. Copyright # 2004 John Wiley & Sons, Ltd. Keywords: -synuclein; amyloid fibril; polyethylene glycol; Ficoll; dextran; crowding agent; conformational change; partially folded intermediate Received 29 November 2003; revised 3 February 2004; accepted 7 February 2004 INTRODUCTION A common property of the interior of all cells is the high concentration of macromolecules present. The typical cell contains  25% protein by volume, of which about 10% forms cytoskeletal filaments and 90% is soluble globular proteins, along with substantial amounts of RNA (rRNAs, mRNAs, tRNAs, small RNAs, etc.) and other biopolymers. Thus, macromolecules occupy about 30% of the cell vo- lume, making that space unavailable to other macromole- cules. This has major thermodynamic and kinetic consequences on the properties of macromolecules present in the cell. These effects can be orders of magnitude different from those in the typical dilute solution used to study proteins in vitro (Ellis, 2001). An idea of the magni- tude of the excluded volume effect can be obtained from the fact that, if 30% of the volume of a cube is filled with macromolecules of a given size, uniformly distributed, then there is virtually no volume available for additional mole- cules of a similar size (Minton, 2001): this leads to highly non linear concentration effects of the crowding agent on reaction equilibria and kinetics. Small molecules, even at very high concentrations, do not cause excluded volume effects, due to their small size. Macromolecular crowding is often taken to be synonymous with excluded volume, however, although the excluded volume effect will always be present, additional factors, such as viscosity, may also be very important, especially in processes such as protein association. There are a number of simple corollaries stemming from the effect of macromolecular crowding that will affect protein aggregation: the free energy of the system will favor the most compact states, e.g. associated states over indivi- dual molecules; decreased water activity will favor de- creased protein solubility and self-association; increased viscosity will lead to decreased diffusion rates and hence decreased kinetics of diffusion-controlled reactions, as in aggregation. The question thus arises as to the net effect of macro- molecular crowding on protein aggregation and fibrillation. Owing to the complexity of the aggregation process, predic tions regarding the effects of macromolecular crowding are not as straightforward as might appear at first sight. Sub- stantial evidence supports the hypothesis that ‘pathological’ or abnormal aggregation (Fink, 1998) arises from a key partially folded intermediate (the amyloidogenic intermedi- ate) (Uversky and Fink, 2003) and that this intermolecular association involves -strand–-strand interactions, but is driven by hydrophobic interactions (Khurana et al., 2002). Such intermediates may have sizable non-polar patches (i.e. contiguous hydrophobic side-chains) on their surface, which lead to hydrophobic interactions between molecules, result- ing in specific intermolecular interactions and aggregation. These hydrophobic patches are absent in the fully unfolded state. Factors that increase the concentration of such inter- mediates will favor aggregation. Several kinetic models for protein aggregation have been put forward (Pallitto and Murphy, 2001; Naiki and Nakakuki, 1996; Come et al., 1993; Lomakin et al., 1996, Copyright # 2004 John Wiley & Sons, Ltd. *Correspondence to: A. L. Fink, Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064, USA. E-mail: enzyme@cats.ucsc.edu Contract/grant sponsor: NIH; contract/grant number: NS39985. Abbreviations used: PD, Parkinson’s disease; PEG, polyethylene glycol; ThT, Thioflavin T.