Folding of beta pep-4 beta-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 identical with 4.184 kJ) from peptide to peptide, are I20V/V22I>I20V>I20L/V22L=beta pep-4 (Delta G(D) of 7.5 kcal/mol)=I20L=I20A>V22I>V22L>V22A for dimer formation and I20V>I20L/V22L>I20L>beta pep-4 (Delta G(T) 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.
Cox
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Department of Biochemistry, Molecular Biology and Biophysics, 6-155 Jackson Hall, University of Minnesota Health Sciences Center, 321 Church Street, Minneapolis, MN 55455, USA.
Arroyo
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Mayo
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D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002942:Circular Dichroism; D019281:Dimerization; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011108:Polymers / Q000737:chemistry; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011506:Proteins / Q000138:chemical synthesis / Q000737:chemistry*; D011994:Recombinant Proteins; D013696:Temperature; D013816:Thermodynamics
D011108:Polymers; D011506:Proteins; D011994:Recombinant Proteins; C111992:betapep-4 protein, recombinant