Unfolding Thermodynamics of the Δ-Domain in the Prohead I Subunit of Phage HK97: Determination by Factor Analysis of Raman Spectra

Abstract An early step in the morphogenesis of the double-stranded DNA (dsDNA) bacteriophage HK97 is the assembly of a precursor shell (prohead I) from 420 copies of a 384-residue subunit (gp5). Although formation of prohead I requires direct participation of gp5 residues 2–103 (Δ-domain), this domain is eliminated by viral protease prior to subsequent shell maturation and DNA packaging. The prohead I Δ-domain is thought to resemble a phage scaffolding protein, by virtue of its highly α-helical secondary structure and a tertiary fold that projects inward from the interior surface of the shell. Here, we employ factor analysis of temperature-dependent Raman spectra to characterize the thermostability of the Δ-domain secondary structure and to quantify the thermodynamic parameters of Δ-domain unfolding. The results are compared for the Δ-domain within the prohead I architecture ( in situ ) and for a recombinantly expressed 111-residue peptide ( in vitro ). We find that the α-helicity (∼ 70%), median melting temperature ( T m  = 58 °C), enthalpy (Δ H m  = 50 ± 5 kcal mol − 1 ), entropy (Δ S m  = 150 ± 10 cal mol − 1 K − 1 ), and average cooperative melting unit (〈 n c 〉 ∼ 3.5) of the in situ Δ-domain are altered in vitro , indicating specific interdomain interactions within prohead I. Thus, the in vitro Δ-domain, despite an enhanced helical secondary structure (∼ 90% α-helix), exhibits diminished thermostability ( T m  = 40 °C; Δ H m  = 27 ± 2 kcal mol − 1 ; Δ S m  = 86 ± 6 cal mol − 1 K − 1 ) and noncooperative unfolding (〈 n c 〉 ∼ 1) vis-a-vis the in situ Δ-domain. Temperature-dependent Raman markers of subunit side chains, particularly those of Phe and Trp residues, also confirm different local interactions for the in situ and in vitro Δ-domains. The present results clarify the key role of the gp5 Δ-domain in prohead I architecture by providing direct evidence of domain structure stabilization and interdomain interactions within the assembled shell.