Structural and Thermodynamic Characterization of T4 Lysozyme ...

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10,000 random coil conformations with an average R g of 38.0 Å, and the second contained 10,000unfolded conformations with residual structure with an average R g of 29.7 Å. The ensemble selectedfrom the pool with residual structure had an average R g of 31.2 Å, in agreement with Guinier analysis,and better described our experimental data than the selected random coil ensemble, which had anaverage R g of 36.1 Å (Figure 5 (b)).Structural Information from Fluorescence Quenching MeasurementsAdditional information on global conformational changes was derived from fluorescence quenchingmeasurements. T4 lysozyme has five methionine residues in close proximity to the three tryptophanresidues present in the C-terminal lobe. The sulfur atom in methionine quenches tryptophan emissionand provides a probe of the methionine-tryptophan separation. Denaturation of T4 lysozyme is usuallyaccompanied by an increase in emission intensity (44), consistent with an increased methioninetryptophandistance. In the seleno-methionine variant of L99A (Se-Met L99A), the methionines arereplaced with seleno-methionines. As selenium and sulfur have differing quenching strengths acomparison of L99A and Se-Met L99A yields additional information on the spatial compactness of theunfolded state of L99A (45).Tryptophan fluorescence measurements were made on Se-Met L99A in pH 3.0, 50 mM glycine,20 mM NaCl and pH 7.0, 50 mM tris, 20 mM NaCl buffers and compared to those of L99A under thesame conditions. The intensity increase accompanying denaturation was more pronounced for Se-MetL99A than for L99A, consistent with the stronger quenching ability of selenium (Figure 6). Figure 7 (a)shows that at each pressure, the centers of spectral mass were similar for L99A and Se-Met L99A in thesame solvent, indicating that these two mutants are structurally similar at any given pressure and that theintroduction of seleno-methionines does not significantly distort the shape of the fluorescence spectra orchange the denaturation behavior.16
Pressure-induced changes to the fluorescence yield of tryptophan or transmission through the solventwere factored out in the ratio of the emission intensities of L99A and Se-Met-L99A, I L99A and I Se-MetL99A ,at the same pressure. The ratios were calculated as follows at each pressureI Se"Met L 99AI L 99A( )NI Se"Met L 99A# ii=1I L 99A (# i )= 1 $ . ( 9 )NThe sum was taken over points in the wavelength range 310-400 nm where the fluorescence signal isstrong. N is the number ! of ratios in the sum (91, in this case). Figure 7 (b) shows a comparison of theseratios at pH 3.0, 50 mM glycine, 20 mM NaCl and pH 7.0, 50 mM Tris, 20 mM NaCl as a function ofdenatured fraction. They are normalized at 0.1 MPa. We see that as denaturation progresses, the ratio isgreater at pH 3.0 than at pH 7.0. This result suggests that the denatured state at pH 7.0 is more compactthan at pH 3.0.Cavity Volume Calculations and Internal WatersThe volumes contained by the external surface and the volumes of internal cavities present in thecrystal structures of the T4 lysozyme mutants were calculated to determine if a correlation existsbetween the volume changes of denaturation extracted from thermodynamic analysis and the volume ofburied cavities. In order to identify and measure buried cavities, crystal structures (PDB accession codes1L90 for L99A, 1QUH for L99G/E108V, 1QS5 for A98L, 1G0P for V149G, and 1L63 for WT*) wereanalyzed with a 1.2 Å probe using MSMS (34). MSMS implements a rolling probe to determine reducedsurfaces, from which the solvent excluded surface can be computed. Two sets of molecular surfaceswere produced for each structure. In the first set of calculations, all crystallographically determinedsolvent particles were manually removed from the PDB files prior to analysis to eliminate false surfacecavities caused by surface water, and to enable detection of hydrated internal cavities. The externalsurface and six to nine cavities were identified for each mutant (Fig. 1). In the second set, internal watermolecules were retained to determine changes in cavity size due to hydration. Four water molecules,Sol-171, 179, 175, and 208, are considered conserved internal solvent molecules in T4 lysozyme (46).17
Page 1: Structural and Thermodynamic Charac
Page 5 and 6: Experimental ProceduresSample and B
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Page 13 and 14: Fluorescence measurements were made
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Page 19 and 20: Unfolding at pH 3.0The volume chang
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Page 23 and 24: BOTTOM QUARTILEIndia, Bangladesh, M
Page 25 and 26: high-pressure crystallography study
Page 27 and 28: esults support the growing view tha
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Page 31 and 32: 27. Glatter, O. (1982) Data Treatme
Page 33 and 34: 43. Bernadó, P., Blanchard, L., Ti
Page 35 and 36: Table 1. Thermodynamic Quantities C
Page 37 and 38: Table 3. Denaturation volume change
Page 39 and 40: andom coil conformers (blue scatter
Page 41 and 42: (a) (b) (c)776655443328673119 1(d)
Page 43 and 44: 365360< λ > (nm)3553503453403350 5
Page 45 and 46: (a)P(r) (a.u.)10.80.60.40.2Iq 22015
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Page 49 and 50: Change in Water Occupancy432100 50
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Page 55: 4. Muchmore, D. C., McIntosh, L. P.

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