Dale E. Tronrud

Lessons from the lysozyme of phage T4

An overview is presented of some of the major insights that have come from studies of the structure, stability, and folding of T4 phage lysozyme. A major purpose of this review is to provide the reader with a complete tabulation of all of the variants that have been characterized, including melting temperatures, crystallographic data, Protein Data Bank access codes, and references to the original literature. The greatest increase in melting temperature (Tm) for any point mutant is 5.1°C for the mutant Ser 117 → Val. This is achieved in part not only by hydrophobic stabilization but also by eliminating an unusually short hydrogen bond of 2.48 Å that apparently has an unfavorable van der Waals contact. Increases in Tm of more than 3–4°C for point mutants are rare, whereas several different types of destabilizing substitutions decrease Tm by 20°C or thereabouts. The energetic cost of cavity creation and its relation to the hydrophobic effect, derived from early studies of “large‐to‐small” mutants in the core of T4 lysozyme, has recently been strongly supported by related studies of the intrinsic membrane protein bacteriorhodopsin. The L99A cavity in the C‐terminal domain of the protein, which readily binds benzene and many other ligands, has been the subject of extensive study. Crystallographic evidence, together with recent NMR analysis, suggest that these ligands are admitted by a conformational change involving Helix F and its neighbors. A total of 43 nonisomorphous crystal forms of different monomeric lysozyme mutants were obtained plus three more for synthetically‐engineered dimers. Among the 43 space groups, P212121 and P21 were observed most frequently, consistent with the prediction of Wukovitz and Yeates.

Introduction to macromolecular refinement

The process of refinement is such a large problem in function minimization that even the computers of today cannot perform the calculations to properly fit X-ray diffraction data. Each of the refinement packages currently under development reduces the difficulty of this problem by utilizing a unique combination of targets, assumptions, and optimization methods. This chapter summarizes the basic methods and underlying assumptions in the commonly used refinement packages. This information can guide the selection of a refinement package that is best suited for a particular refinement project.

Refinement of the Structure of a Water-Soluble Antenna Complex from Green Photosynthetic Bacteria by Incorporation of the Chemically Determined Amino Acid Sequence

The green photosynthetic bacterium Prosthecochloris aestuarii contains a water-soluble chlorophyllcontaining protein that forms part of the lightgathering antenna complex (Olson, 1978). The crystallographic structure determination of the protein revealed it to be a trimer of three identical subunits, each containing seven bacteriocNorophyll a (Bchla) molecules (Fenna and Matthews, 1975; Matthews and Fenna, 1980). The three-dimensional crystal structure of the protein has been refined (Tronrud et al., 1986), permitting the overall molecular architecture of the protein and the conformations of the Bchls to be defined fairly reliably. At the time, however, the amino acid sequence of the protein was unknown, so it was necessary to infer a tentative amino acid sequence from the crystallographic data alone. This task is not an easy one. First, the lack of an independently determined amino acid sequence makes it difficult to interpret, with confidence, the electron density maps used to define the structure of the protein. Second, &en if the overall conformation of the protein is interpreted correctly, it is difficult to identlfy individual amino acids and to distinguish essentially isostructural pairs such as threonine and valine, glutamate and glutamine, and aspartate and asparagine. Subsequently, however, Fenna and co-workers (Daurat-Larroque et aL, 1986) determined the amino acid sequence of the Bchla protein, making it

Evaluation at atomic resolution of the role of strain in destabilizing the temperature-sensitive T4 lysozyme mutant Arg 96 --> His.

Mutant R96H is a classic temperature‐sensitive mutant of bacteriophage T4 lysozyme. It was in fact the first variant of the protein to be characterized structurally. Subsequently, it has been studied extensively by a variety of experimental and computational techniques, but the reasons for the loss of stability of the mutant protein remain controversial. In the crystallographic refinement of the mutant structure at 1.9 Å resolution one of the bond angles at the site of substitution appeared to be distorted by about 11°, and it was suggested that this steric strain was one of the major factors in destabilizing the mutant. Different computationally‐derived models of the mutant structure, however, did not show such distortion. To determine the geometry at the site of mutation more reliably, we have extended the resolution of the data and refined the wildtype (WT) and mutant structures to be better than 1.1 Å resolution. The high‐resolution refinement of the structure of R96H does not support the bond angle distortion seen in the 1.9 Å structure determination. At the same time, it does confirm other manifestations of strain seen previously including an unusual rotameric state for His96 with distorted hydrogen bonding. The rotamer strain has been estimated as about 0.8 kcal/mol, which is about 25% of the overall reduction in stability of the mutant. Because of concern that contacts from a neighboring molecule in the crystal might influence the geometry at the site of mutation we also constructed and analyzed supplemental mutant structures in which this crystal contact was eliminated. High‐resolution refinement shows that the crystal contacts have essentially no effect on the conformation of Arg96 in WT or on His96 in the R96H mutant.

Sorting the chaff from the wheat at the PDB

There is no dispute that the overwhelming majority of the 50,000 structures in the Protein Data Bank are essentially correct (i.e., lacking major experimental error). Nevertheless, there have been and there continue to be reports of protein structures which are seriously in error. A number of these are listed in Table I.

Crystallographic and Genetic Approaches Toward the Design of Proteins of Enhanced Thermostability

The advent of directed mutagenesis has made it possible to alter protein structures at will. For the first time it is possible to design and to introduce modifications into a protein that are intended to change its behavior in predictable ways.

Representing φ/ψ-dependency of a variable as a continuous function

In work to develop conformation dependant libraries (CDL) [1] for peptide geometry it is desirable to describe the variation of target values for main chain bond angles as a continuous function of φ and ψ. In our previous work [2, 3] the Ramachandran plot was broken into 10°x10° tiles and the bond angle values for real structures assigned to the appropriate tiles. The variation in average values across the tiles was smoothed with kernel density averaging to generate a table of target values for that bond angle. In protein structures the conformations of residues are not uniformly distributed, so some tiles may contain a great many samples while others contain none. The “unpopular” tiles must be treated as special cases and the transition between the populated regions and the unpopulated creates difficulties. In addition, any target value for a bond angle that changes quickly relative to the 10° spacing of the tiles will become a “stepped pyramid” and not a smooth hillside. Specifically, with sparse datasets, such as occur in our current work to develop CDLs for cis-peptide units, the number of sample points in a residue category range from 796 to 6 and their density in φ/ψ space also varies. We believed that the level of detail of the CDL should be contingent on the nature of the sample sets, but optimizing the predictive power of the library by changing the size of the tiles, the kernel density averaging parameter κ, and the significance cutoff proved impossible due to the high variability of the target function caused by the discontinuities of the form of the library.