Unfolding thermodynamics of the Delta-domain in the prohead I subunit of phage HK97: determination by factor analysis of Raman spectra.

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 (Delta-domain), this domain is eliminated by viral protease prior to subsequent shell maturation and DNA packaging. The prohead I Delta-domain is thought to resemble a phage scaffolding protein, by virtue of its highly alpha-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 Delta-domain secondary structure and to quantify the thermodynamic parameters of Delta-domain unfolding. The results are compared for the Delta-domain within the prohead I architecture (in situ) and for a recombinantly expressed 111-residue peptide (in vitro). We find that the alpha-helicity (approximately 70%), median melting temperature (T(m)=58 degrees C), enthalpy (DeltaH(m)=50+/-5 kcal mol(-1)), entropy (DeltaS(m)=150+/-10 cal mol(-1) K(-1)), and average cooperative melting unit (n(c) approximately 3.5) of the in situ Delta-domain are altered in vitro, indicating specific interdomain interactions within prohead I. Thus, the in vitro Delta-domain, despite an enhanced helical secondary structure ( approximately 90% alpha-helix), exhibits diminished thermostability (T(m)=40 degrees C; DeltaH(m)=27+/-2 kcal mol(-1); DeltaS(m)=86+/-6 cal mol(-1) K(-1)) and noncooperative unfolding ( approximately 1) vis-à-vis the in situ Delta-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 Delta-domains. The present results clarify the key role of the gp5 Delta-domain in prohead I architecture by providing direct evidence of domain structure stabilization and interdomain interactions within the assembled shell.

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