Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 Oct 21;47(42):11097-109.
doi: 10.1021/bi801287m. Epub 2008 Sep 25.

Structural and thermodynamic characterization of T4 lysozyme mutants and the contribution of internal cavities to pressure denaturation

Nozomi Ando  1 , Buz Barstow, Walter A Baase, Andrew Fields, Brian W Matthews, Sol M Gruner
Affiliations

Structural and thermodynamic characterization of T4 lysozyme mutants and the contribution of internal cavities to pressure denaturation

Nozomi Ando et al. Biochemistry. .

Abstract

Using small-angle X-ray scattering (SAXS) and tryptophan fluorescence spectroscopy, we have identified multiple compact denatured states of a series of T4 lysozyme mutants that are stabilized by high pressures. Recent studies imply that the mechanism of pressure denaturation is the penetration of water into the protein rather than the transfer of hydrophobic residues into water. To investigate water penetration and the volume change associated with pressure denaturation, we studied the solution behavior of four T4 lysozyme mutants having different cavity volumes at low and neutral pH up to a pressure of 400 MPa (0.1 MPa = 0.9869 atm). At low pH, L99A T4 lysozyme expanded from a compact folded state to a partially unfolded state with a corresponding change in radius of gyration from 17 to 32 A. The volume change upon denaturation correlated well with the total cavity volume, indicating that all of the molecule's major cavities are hydrated with pressure. As a direct comparison to high-pressure crystal structures of L99A T4 lysozyme solved at neutral pH [Collins, M. D., Hummer, G., Quillin, M. L., Matthews, B. W., and Gruner, S. M. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 16668-16671], pressure denaturation of L99A and the structurally similar L99G/E108V mutant was studied at neutral pH. The pressure-denatured state at neutral pH is even more compact than at low pH, and the small volume changes associated with denaturation suggest that the preferential filling of large cavities is responsible for the compactness of the pressure-denatured state. These results confirm that pressure denaturation is characteristically distinct from thermal or chemical denaturation.

PubMed Disclaimer

Figures

Figure 1
Figure 1
T4 lysozyme structures shown from the same perspective. The external surface and buried cavities (shown in magenta) of (a) WT* (1L63), (b) L99A (1L90), (c) L99G/E108V (1QUH), (d) A98L (1QS5), and (e) V149G (1G0P) identified with a 1.2 Å probe in MSMS (34) with all internal solvent molecules removed. Cavities are identified by numbers 1 - 10 (refer to Table 2). (f) Cartoon representation of WT* T4 lysozyme. The C-terminal lobe is on the top side.
Figure 2
Figure 2
Pressure denaturation of T4 lysozyme mutants in pH 3.0 50 mM glycine 20 mM NaCl (diamond) and 100 mM NaCl (circle) buffers monitored at 16 °C by tryptophan fluorescence spectroscopy. (a) L99G/E108V (open) and L99A (closed). (b) V149G (open) and A98L (closed).
Figure 3
Figure 3
Pressure denaturation of L99G/E108V (diamond), L99A (circle), and WT* (plus) T4 lysozyme in pH 7.0 50 mM Tris HCl 20 mM NaCl buffer monitored at 24 °C by tryptophan fluorescence spectroscopy.
Figure 4
Figure 4
(a) Radius of gyration, Rg, as a function of pressure for 10 g/l L99A in 50 mM glycine 100 mM NaCl pH 3.0 buffer at room temperature. Rg was determined by a Guinier fit to the qRg < 1.3 region of scattering profiles taken at each pressure. The error bars are larger at high pressure because only one exposure was taken at each pressure. At lower pressure, the sample was less susceptible to radiation damage-induced aggregation and multiple exposures were taken at each pressure, which enabled averaging of images. A two-state thermodynamic fit is shown (solid line) to guide the eye. (b) The zero-angle scattering intensity of L99A (circle) and the WT* (diamond) at pH 3.0. WT* does not denature below 300 MPa.
Figure 5
Figure 5
(a) Pair distance distribution functions of native (28 MPa, solid line) and denatured (300 MPa, dotted line) L99A T4 lysozyme at pH 3.0 obtained with GNOM (28). Inset: Kratky representation of the 28 MPa (1) and 300 MPa data (2). (b) The scattering profile of L99A at 300 MPa (black) was examined with the Ensemble Optimization Method (30). An ensemble of unfolded conformers with residual structure (red scattering profile) better described the experimental data compared to an ensemble of random coil conformers (blue scattering profile). The distribution of Rg in the first ensemble is shown in the inset. (c) Side and top views of the crystal structure of L99A (right) and a representative low-resolution structure at 28 MPa obtained with GASBOR (29) (left) show good agreement. (d) GASBOR models that fit well to 300 MPa data were extended.
Figure 6
Figure 6
Tryptophan fluorescence spectra of L99A (color) and Se-Met L99A (black) in 50 mM glycine 20 mM NaCl pH 3.0 16 °C with increasing pressure (direction indicated by arrow). The emission intensities were normalized at high pressure (200 MPa) to emphasize the pressure-induced intensity increase of Se-Met L99A compared with L99A. Inset: Spectra of L99A (red) and Se-Met L99A (black) at low pressure (25 MPa) scaled by a constant factor.
Figure 7
Figure 7
Pressure denaturation of L99A and Se-Met L99A in 50 mM glycine 20 mM NaCl pH 3.0 16 °C and 50 mM Tris HCl 20 mM NaCl pH 7.0 24 °C. (a) Centers of spectral mass of L99A (open) and Se-Met L99A (closed) at pH 3.0 (diamond) and pH 7.0 (circle). L99A and Se-Met L99A show similar denaturation curves, indicative of structural similarity at each pressure. (b) Normalized intensity ratios of Se-Met L99A and L99A emission at pH 3.0 (closed diamond) and pH 7.0 (open circle). At pH 7.0, the ratio shows little change with pressure, while at pH 3.0, there is a large increase. This indicates a greater separation between tryptophan and seleno-methionine residues in the denatured state at pH 3.0 compared with pH 7.0, suggesting that the pH 3.0 denatured state is more unfolded than at pH 7.0.
Figure 8
Figure 8
The volume changes of denaturation (see Table 1) of L99G/E108V, L99A, V149G, and A98L T4 lysozyme in pH 3.0 buffer, 16 °C at 20 mM NaCl (open diamond) and 100 mM NaCl (closed circle) compared to the total cavity volumes calculated from crystal structures using two methods. (a) No correlation is apparent when full occupancy of all crystallographically determined internal solvent-biding sites was assumed in the calculation of cavity volumes. (b) The large cavity of L99G/E108V was assumed to be empty, while full occupancy was assumed for all other internal solvent molecules. The volume changes of denaturation correlate with the total cavity volumes.
Figure 9
Figure 9
Comparison of change in water occupancy of L99A at neutral pH determined by X-ray crystallography (diamond, reprinted with permission from Collins, et al. (1)) and fluorescence spectroscopy (circle). A two-state (0 or 3 water molecules) thermodynamic fit to the data is shown on each data set with a fixed volume change of 90 Å3 (volume of three water molecules in bulk). The fit to the crystallography data is shifted to lower pressure by 82 MPa relative to the fluorescence data. This stability difference may be due to subtle differences between structure and dynamics of the T4 lysozyme molecule in solution and in the crystal. The change in water occupancy of WT* determined by fluorescence is shown for reference (cross).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Collins MD, Hummer G, Quillin ML, Matthews BW, Gruner SM. Cooperative water filling of a nonpolar protein cavity observed by high-pressure crystallography and simulation. Proc. Nat. Acad. Sci U.S.A. 2005;102:16668–16671. [published erratum appears in (2006) Proc. Nat. Acad. Sci U.S.A. 103, 4793] - PMC - PubMed
    1. Dill KA, Shortle D. Denatured states of proteins. Annu. Rev. Biochem. 1991;60:795–825. - PubMed
    1. Kauzmann W. Denaturation of proteins and enzymes. In: McElroy WD, Glass B, editors. The mechanism of enzyme action. Johns Hopkins Press; Baltimore, MD: 1954. pp. 70–110.
    1. Kauzmann W. Some factors in the interpretation of protein denaturation. Adv. Protein Chem. 1959;14:1–63. - PubMed
    1. Dill KA. Dominant forces in protein folding. Biochemistry. 1990;29:7133–7155. - PubMed

Publication types