Ž . Biochimica et Biophysica Acta 1382 1998 257–265 Measurement of peptide aggregation with pulsed-field gradient nuclear magnetic resonance spectroscopy Shawn L. Mansfield, Dimuthu A. Jayawickrama, Jeffery S. Timmons, Cynthia K. Larive ) Department of Chemistry, UniÕersity of Kansas, Lawrence, KS 66045, USA Received 26 June 1997; accepted 4 September 1997 Abstract Interactions between hydrophobic patches in proteins are often a driving force for denaturation and aggregation. The ŽŽ .. aggregation of the b-amyloid peptide fragment, VHHQKLVFFAEDVGSNK b 12–28 , has been investigated in aqueous solution at low pH. This peptide contains a central hydrophobic patch spanning residues 17–21. Diffusion coefficients measured with pulsed-field gradient NMR as a function of peptide solution concentration were used to assess the extent of aggregation. Following the hypothesis that hydrophobic interactions are an important driving force in the aggregation of this Ž . w 19,20 x Ž . peptide at low pH, a non-aggregating analog of the b 12–28 peptide, Gly b 12–28 was synthesized. In the w 19,20 x Ž . Gly b 12–28 peptide, the replacement of the two phenylalanine residues disrupts the hydrophobic interactions which Ž . w 19,20 x Ž . drive the aggregation of b 12–28 . The diffusion coefficient of the Gly b 12–28 peptide is invariant over the Ž . concentration range studied and provides a good estimate of the monomeric diffusion coefficient of b 12–28 . A second peptide analog was synthesized in which the phenylalanine at position 20 was replaced with a cysteine residue. The Žw 20 x Ž .. disulfide-linked dimer, Cys b 12–28 , was formed upon air oxidation of this peptide. The diffusion coefficient of the 2 Žw 20 x Ž .. Ž . Cys b 12–28 peptide was measured and used to estimate the diffusion coefficient of the b 12–28 dimer. Using the 2 monomeric and dimeric diffusion coefficients measured for the glycine and cysteine analogs, the concentration dependence Ž . of the b 12–28 diffusion coefficient was found to be consistent with a monomer–dimer aggregation model. q 1998 Elsevier Science B.V. Keywords: Pulsed-field gradient NMR; Diffusion coefficient; Aggregation, Ab peptide fragment; Hydrophobic interaction 1. Introduction Ž . The b-amyloid Ab peptide is a 40–43 residue peptide that has been implicated in the plaque forma- tion associated with Alzheimer’s disease. Several studies of Ab and related peptide fragments and analogs have focused on the central hydrophobic w x region of the peptide spanning residues 17–21 1–5 . ) Corresponding author. Fax: q1 785 864-5396; E-mail: CLarive@CaC03.chem.ukans.edu Recently, Esler and co-workers reported that substitu- tion of phenylalanine 19 in the plaque-competent Ž . w 19 x Ž . b 10–35 with a threonine, Thr b 10–35 , trans- wx forms it into a plaque-incompetent peptide 5 . There- fore, this region of the peptide is important in the biological and in vitro physical chemical behavior of the Ab peptide and its analogs. The Ab fragment, VHHQKLVFFAEDVGSNK Ž Ž .. b 12–28 , is a good model compound for studying peptide aggregation because of the biological activity and the propensity for aggregation of the parent 0167-4838r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. Ž . PII S0167-4838 97 00162-3