For the designed peptide 33mer, βpep-4, formation of β-sheet structure [Ilyina, Roongta and Mayo (1997) Biochemistry 36, 5245–5250] is thermodynamically linked to self-association. Dimers and tetramers are stabilized by interactions between hydrophobic residues lying on the hydrophobic faces of the amphipathic monomer subunits. The present study investigates the effects on folding and self-association of the substitution of two key hydrophobic residues (Ile20 and Val22) at the β-sheet sandwich interface of βpep-4. Single-site (I20L, I20V, I20A, V22L, V22I and V22A; where I20L corresponds to the substitution of Ile20 with leucine etc.) and double-site (I20L/V22L and I20V/V22I) variants have been investigated. Like parent βpep-4, all variants can form dimers and tetramers. NOESY data indicate that the overall β-sheet fold and intersubunit β-strand alignments are the same in all variant tetramers. CD data for all variants indicate mostly β-sheet character in dimers and random coil character in monomers. Only for the V22I variant is the β-sheet fold stabilized in the monomer state. Pulse-field gradient NMR-derived diffusion coefficients, measured as a function of peptide concentration, provide a means for deriving the distribution of monomer, dimer and tetramer states and, therefore, equilibrium association constants. Relative thermodynamic stabilities, which vary no more than approx. 0.5kcal/mol (where 1kcal≡4.184kJ) from peptide to peptide, are I20V/V22I>I20V>I20L/V22L = βpep-4 (ΔGD of 7.5kcal/mol) = I20L = I20A>V22I>V22L>V22A for dimer formation and I20V>I20L/V22L>I20L>βpep-4 (ΔGT of 6kcal/mol)>V22I>I20V/V22I>V22L>I20A>V22A for tetramer formation. For the most part, dimer and/or tetramer stabilities are enhanced by the presence of valine and leucine and are attenuated by the presence of isoleucine and alanine.

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