Michael L. Quillin

Accurate calculation of the density of proteins

On the basis of theoretical calculations, Andersson & Hovmöller have recently suggested that the long-established value of 1.35 g cm(-3) for the mean density of proteins should be revised to 1.22 g cm(-3) [Andersson & Hovmöller (2000), Acta Cryst. D56, 789-790]. To substantiate their assertion, these authors used the Voronoi algorithm to calculate the mean atomic volume for 30 representative protein structures. The Voronoi procedure requires that atoms of interest be bounded on all sides by other atoms. Volume calculations for surface atoms that are not surrounded or are only sparsely surrounded by other atoms either are not possible or may be unreliable. In an attempt to circumvent this problem, Andersson & Hovmöller rejected atoms with calculated volumes that were indeterminate or were greater than 50 A(3). In the present study, it is shown that this criterion is not sufficiently restrictive to ensure accurate volume determinations. When only strictly buried atoms are included in the volume calculations using the Voronoi algorithm, the mean density is found to be 1.47 +/- 0.05 g cm(-3). In addition, an alternate procedure based on the Connolly algorithm that permits all protein atoms to be included in volume calculations gives 1.43 +/- 0.03 g cm(-3) for the mean density of the same set of proteins. The latter two calculated values are mutually consistent and are in better agreement with the experimental value.

Structural and thermodynamic analysis of the binding of solvent at internal sites in T4 lysozyme

To investigate the structural and thermodynamic basis of the binding of solvent at internal sites within proteins a number of mutations were constructed in T4 lysozyme. Some of these were designed to introduce new solvent‐binding sites. Others were intended to displace solvent from preexisting sites. In one case Val‐149 was replaced with alanine, serine, cysteine, threonine, isoleucine, and glycine. Crystallographic analysis shows that, with the exception of isoleucine, each of these substitutions results in the binding of solvent at a polar site that is sterically blocked in the wild‐type enzyme. Mutations designed to perturb or displace a solvent molecule present in the native enzyme included the replacement of Thr‐152 with alanine, serine, cysteine, valine, and isoleucine. Although the solvent molecule was moved in some cases by up to 1.7Å, in no case was it completely removed from the folded protein. The results suggest that hydrogen bonds from the protein to bound solvent are energy neutral. The binding of solvent to internal sites within proteins also appears to be energy neutral except insofar as the bound solvent may prevent a loss of energy due to potential hydrogen bonding groups that would otherwise be unsatisfied. The introduction of a solvent‐binding site appears to require not only a cavity to accommodate the water molecule but also the presence of polar groups to help satisfy its hydrogen‐bonding potential. It may be easier to design a site to accommodate two or more water molecules rather than one as the solvent molecules can then hydrogen‐bond to each other. For similar reasons it is often difficult to design a point mutation that will displace a single solvent molecule from the core of a protein.

Use of experimental crystallographic phases to examine the hydration of polar and nonpolar cavities in T4 lysozyme

There is conflicting evidence as to whether cavities in proteins that are nonpolar and large enough to accommodate solvent are empty or are occupied by disordered water molecules. Here, we use multiple-wavelength x-ray data collected from crystals of the selenomethionine-substituted L99A/M102L mutant of T4 lysozyme to obtain a high-resolution electron density map free of bias that is unavoidably associated with conventional model-based structure determination and refinement. The mutant, L99A/M102L, has four cavities, two being polar in character and the other two nonpolar. Cavity 1 (polar, volume 45.2 Å3) was expected to contain two well ordered water molecules, and this is confirmed in the experimental electron density map. Likewise, cavity 2 (polar, 16.9 Å3) is confirmed to contain a single water molecule. Cavity 3 (nonpolar, 21.4 Å3) was seen to be empty in conventional x-ray refinement, and this is confirmed in the experimental map. Unexpectedly, however, cavity 4 (nonpolar, volume 133.5 Å3) was seen to contain diffuse electron density equivalent to ≈1.5 water molecules. Although cavity 4 is largely nonpolar, it does have some polar character, and this apparently contributes to the presence of solvent. The cavity is large enough to accommodate four to five water molecules, and it appears that a hydrogen-bonded chain of three or more solvent molecules could occupy the cavity at a given time. The results are consistent with theoretical predictions that cavities in proteins that are strictly nonpolar will not contain solvent until the volume is large enough to permit mutually satisfying water–water hydrogen bonds.

Determination of solvent content in cavities in IL-1beta using experimentally phased electron density

The extent to which water is present within apolar cavities in proteins remains unclear. In the case of interleukin-1β (IL-1β), four independent structures solved by x-ray crystallography indicate that water is not present in the central apolar cavity. In contrast, results from NMR spectroscopy suggest that water has high occupancy within the cavity but is positionally disordered, making it undetectable by standard crystallographic methods. A theoretically based crystallographic-phase refinement technique also suggested that there was the equivalent of two fully occupied water molecules within the apolar cavity. To resolve these discrepancies we sought to obtain an experimentally phased electron density map that was free of possible bias caused by mathematical modeling of the protein or the solvent. By combining native diffraction data with multiple wavelength anomalous data from a platinum derivative, accurate phases were obtained. Using these experimental phases, we estimate that occupancy of the apolar cavity in IL-1β by solvent is close or equal to zero. Polar cavities in the protein that contain ordered solvent molecules serve as internal controls.

Multiple methionine substitutions are tolerated in T4 lysozyme and have coupled effects on folding and stability

In order to further explore the tolerance of proteins to amino acid substitutions within the interior, a series of core residues was replaced by methionine within the C-terminal domain of T4 lysozyme. By replacing leucine, isoleucine, valine and phenylalanine residues a total of 10 methionines could be introduced, which corresponds to a third of the residues that are buried in this domain. As more methionines are incorporated the protein gradually loses stability. This is attributed in part to a reduction in hydrophobic stabilization, in part to the increased entropic cost of localizing the long, flexible methionine sidechains, and in part to steric clashes. The changes in structure of the mutants relative to the wildtype protein are modest but tend to increase in an additive fashion as more methionines are included. In the most extreme case, namely the 10-methionine mutant, much of the C-terminal domain remains quite similar to wildtype (root-mean-square backbone shifts of 0.56 A), while the F and G helices undergo rotations of approximately 20 degrees and center-of-mass shifts of approximately 1.4 A. For up to six methionine substitutions the changes in stability are additive. Beyond this point, however, the multiple mutants are somewhat more stable than suggested from the sum of their constituents, especially for those including the replacement Val111-->Met. This is interpreted in terms of the larger structural changes associated with this substitution. The substituted sidechains in the mutant structures have somewhat higher crystallographic thermal factors than their counterparts in WT*. Nevertheless, the interiors of the mutant proteins retain a well-defined structure with little suggestion of molten-globule characteristics. Lysozymes in which selenomethionine has been incorporated rather than methionine tend to have increased stability. At the same time they also fold faster. This provides further evidence that, at the rate-limiting step in folding, the structure of the C-terminal domain of T4 lysozyme is similar to that of the fully folded protein.

Selling candles in a post-Edison world: phasing with noble gases bound within engineered sites

The utility of noble gases for phase determination has been limited by the lack of naturally occurring binding sites in proteins. Wild-type T4 lysozyme contains one such binding site. By mutating large hydrophobic residues to alanine, additional noble-gas binding sites have been successfully introduced into this protein. Using data from xenon derivatives of the wild type, two single mutants and the corresponding double mutant, experimental phases for T4 lysozyme have been determined using standard multiple isomorphous replacement (MIR) techniques. These phases, which were obtained from room-temperature data collected on a rotating-anode source, are comparable in quality with phases calculated using selenomethionine-based multiwavelength anomalous dispersion (MAD) methods on frozen crystals at a synchrotron. In addition, this method of introducing noble-gas binding sites near specific residues should provide useful information for determining the register of amino acids within electron-density maps and the positions of molecules within the unit cell.