Folding of βpep-4 β-sheet sandwich dimers and tetramers is influenced by aliphatic hydrophobic residues at the intersubunit interface

For the designed peptide 33mer, beta pep-4, formation of beta -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 (Ile(20) and Val(22)) at the beta -sheet sandwich interface of beta pep-4. Single-site (I20L, I20V, I20A, V22L, V22I and V22A; where I20L corresponds to the substitution of Ile(20) with leucine etc.) and double-site (I20L/V22L and I20V/V22I) variants have been investigated. Like parent,beta pep-4, all variants can form dimers and tetramers. NOESY data indicate that the overall beta -sheet fold and intersubunit beta -strand alignments are the same in all variant tetramers. CD data for all variants indicate mostly beta -sheet character in dimers and random coil character in monomers. Only for the V22I variant is the beta -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. 0.5 kcal/mol (where 1 kcal equivalent to 4.184 kJ) from peptide to peptide, are I20V/V22I > I20V > I20L/V22L = beta pep-4 (DeltaG(D) of 7.5 kcal/mol) = I20L = I20A > V22I > V22L > V22A for dimer formation and I20V > I20L/V22L > I20L > beta pep-4 (AG, of 6 kcal/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.