pubmed.ncbi.nlm.nih.gov

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

  • ️Thu Jan 01 2009

Comparative Study

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

Daniel Nemecek et al. J Mol Biol. 2009.

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 (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 (<n(c)> 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.

PubMed Disclaimer

Figures

Figure 1
Figure 1

(a) Sedimentation velocity of the in vitro Δ-domain. (Top) Absorbance at 280 nm as a function of radial position during Δ-domain (85 μM) sedimentation at 4 °C. Data were acquired in 90 s intervals. For clarity, the results for 15 min intervals are shown (colored circles). Sedimentation boundaries were fitted by the Lamm equation (black lines). Corresponding residuals are shown in the lower field. (Bottom) Fitted distributions of the apparent sedimentation coefficient (left) and molecular mass (right) reveal a single species of s* = 0.8 (corresponding to s020,w = 1.3) and 12 kDa, consistent with an elongated monomer. (b) Sedimentation equilibrium of the in vitro Δ-domain. Samples (87, 67 and 58 μM) at 4 °C were spun to equilibrium at 35,000 (red circles), 40,000 (green triangles) and 45,000 rpm (blue squares). The profiles were acquired at 280 nm and fitted with eq. 5 to determine the molecular mass of 12 ± 1 kDa.

Figure 2
Figure 2

(a) Temperature-dependence of the Raman spectrum of the in vitro Δ-domain. The spectra obtained at 10 and 90 °C and the difference spectra computed for each 10 °C interval between 10 and 80 °C are shown, as labeled. The major spectral changes, which correspond to Δ-domain unfolding, occur for 30–40 and 40–50 °C intervals. Raman intensities were normalized using the integrated intensity of the amide I band (see text). (b) Factor analysis of the temperature-dependent Raman spectra of the in vitro Δ-domain. Singular values (plotted on a logarithmic scale) and residual errors reveal that three factors are sufficient to describe all spectral changes associated with Δ-domain unfolding (factor dimension M = 3). The three factors were fitted to a two-state model using eqs. 3 and 4. The red lines show the precision of the fitted coefficients Vi1-Vi3.

Figure 3
Figure 3

(a) Plots of the mole fractions of folded and unfolded states of the in vitro Δ-domain as a function of temperature. The fractions were determined from the fitted thermodynamic parameters using eqs. 3 and 4 (lines) and by spectral decomposition of the temperature-dependent Raman spectra of the in vitro Δ-domain into the spectra determined for folded and unfolded states of the in vitro Δ-domain (circles and triangles, respectively). (b) Plots of the mole fractions of folded and unfolded states of the in situ Δ-domain as a function of temperature. The fractions were determined from the fitted thermodynamic parameters using eqs. 3 and 4 (lines) and by spectral decomposition of the temperature-dependent Raman spectra of the in situ Δ-domain into the spectra determined for folded and unfolded states of the in situ Δ-domain (circles and triangles, respectively). (c) Comparison of the Raman spectra of the folded Δ-domain in situ (top) and in vitro (middle). The difference spectrum (bottom) reveals changes in secondary structure and local environments of Trp and Phe side chains of the in situ Δ-domain (see text). (d) Comparison of the Raman spectra of the unfolded Δ-domain in situ (top) and in vitro (middle). The difference spectrum (bottom) shows that changes in secondary structure and Trp and Phe local environments are smaller than those occurring for the folded Δ-domain (cf. bottom trace of panel (c)).

Figure 4
Figure 4

(a) Temperature-dependence of the Raman spectrum of prohead I. The spectra obtained at 10 and 92 °C and the difference spectra computed for the indicated intervals between 10 and 92 °C are shown, as labeled. The largest spectral change, which corresponds to prohead I disassembly, occurs above 83 °C. The other major spectral transition, which occurs primarily between 50 and 60 °C, corresponds to Δ-domain unfolding within the prohead I assembly. Raman intensities were normalized to the integrated intensity of the amide I band. (The intensity of the tyrosine marker at 644 cm−1 is also invariant to temperature.) (b) Temperature-dependence of the Raman spectrum of prohead II, showing a single transition above 83 °C, which corresponds to prohead II disassembly.

Figure 5
Figure 5

(a) Temperature-dependence of the Raman spectrum of the in situ Δ-domain. The spectra obtained at 10 and 83 °C and the difference spectra computed for each 10 °C interval between 10 and 80 °C are shown, as labeled. The major spectral changes, which correspond to Δ-domain unfolding, occur principally in the 50–60 °C interval. Raman intensities were normalized using the integrated intensity of the amide I band. (b) Factor analysis of the temperature-dependent Raman spectra of the in situ Δ-domain. Singular values (plotted on a logarithmic scale) and residual errors reveal a factor dimension M = 4. The four factors were fitted to a two-state model using eqs. 3 and 4. The red lines show the precision of the fitted coefficients Vi1-Vi4.

Figure 6
Figure 6

A comparison of thermally induced perturbations to Δ-domain secondary structure in vitro (top) and in situ (bottom). The models incorporate results of the Raman amide I band analyses and the sizes of the cooperative units (〈nc〉) determined from sedimentation and thermodynamic data (see text). A key feature is that at physiological temperature (37 °C) the structural organization of the in situ Δ-domain is greater than that of the in vitro Δ-domain. In particular, each chain of the in situ trimer retains full α-helicity, whereas the in vitro monomer is partially unfolded.

Similar articles

Cited by

References

    1. Simon LD. Infection of Escherichia coli by T2 and T4 bacteriophages as seen in the electron microscope: T4 head morphogenesis. Proc Natl Acad Sci U S A. 1972;69:907–911. - PMC - PubMed
    1. Trus BL, Booy FP, Newcomb WW, Brown JC, Homa FL, Thomsen DR, Steven AC. The herpes simplex virus procapsid: structure, conformational changes upon maturation, and roles of the triplex proteins VP19c and VP23 in assembly. J Mol Biol. 1996;263:447–462. - PubMed
    1. Butcher SJ, Dokland T, Ojala PM, Bamford DH, Fuller SD. Intermediates in the assembly pathway of the double-stranded RNA virus ϕ6. EMBO J. 1997;16:4477–4487. - PMC - PubMed
    1. King J, Chiu W. The Procapsid to Capsid Transition in Double-stranded DNA Bacteriophages. In: Chiu W, Burnett RM, Garcea R, editors. Structural Biology of Viruses. Oxford University Press; New York: 1997. pp. 288–311.
    1. Turner BG, Summers MF. Structural biology of HIV. J Mol Biol. 1999;285:1–32. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources