Biomedical subjects

Walter A Baase

Publications and source records attributed to Walter A Baase.

5 recordsLinked to original sources

PubMed2003

Repacking the Core of T4 lysozyme by automated design.

Automated protein redesign, as implemented in the program ORBIT, was used to redesign the core of phage T4 lysozyme. A total of 26 buried or partially buried sites in the C-terminal domain were allowed to vary both their sequence and side-chain conformation while the backbone and non-selected side-chains remained fixed. A variant with seven substitutions ("Core-7") was identified as having the most favorable energy. The redesign experiment was repeated with a penalty for the presence of methionine residues. In this case the redesigned protein ("Core-10") had ten amino acid changes. The two designed proteins, as well as the constituent single mutants, and several single-site revertants were over-expressed in Escherichia coli, purified, and subjected to crystallographic and thermal analyses. The thermodynamic and structural data show that some repacking was achieved although neither redesigned protein was more stable than the wild-type protein. The use of the methionine penalty was shown to be effective. Several of the side-chain rotamers in the predicted structure of Core-10 differ from those observed. Rather than changing to new rotamers predicted by the design process, side-chains tend to maintain conformations similar to those seen in the native molecule. In contrast, parts of the backbone change by up to 2.8A relative to both the designed structure and wild-type. Water molecules that are present within the lysozyme molecule were removed during the design process. In the redesigned protein the resultant cavities were, to some degree, re-occupied by side-chain atoms. In the observed structure, however, water molecules were still bound at or near their original sites. This suggests that it may be preferable to leave such water molecules in place during the design procedure. The results emphasize the specificity of the packing that occurs within the core of a typical protein. While point substitutions within the core are tolerated they almost always result in a loss of stability. Likewise, combinations of substitutions may also be tolerated but usually destabilize the protein. Experience with T4 lysozyme suggests that a general core repacking methodology with retention or enhancement of stability may be difficult to achieve without provision for shifts in the backbone.

Automation↗

PubMed2003

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.

Amino Acid Substitution↗

PubMed2002

A helix initiation signal in T4 lysozyme identified by polyalanine mutagenesis.

To better understand the relation between sequence and structure, and in an attempt to simplify the protein folding problem, a series of alanine substitutions was introduced into bacteriophage T4 lysozyme. In contrast to previous studies in this system, which were restricted to single alpha-helices, the present analysis included a helix-turn-helix region, a loop-helix region, and two alpha-helices that were well separated in the three-dimensional structure. It was shown previously that T4 lysozyme is very tolerant of alanine substitutions within alpha-helices, especially at solvent-exposed sites. The present study shows that the protein is also tolerant of such substitutions in turn and loop regions, although less than in helices. The results confirm that the structural information in the amino acid sequence is highly redundant. For example, the protein with the sequence 127AAAAAALAAAAWAAA141 folds normally, has melting temperature only 0.8 degrees C lower than wildtype, and has a crystal structure that is also very similar to wildtype. Polyalanine substitutions within turns or loops can, however, lead to differences in structure and in folding. In one example the triple substitution K35A/S36A/P37A caused this region of the molecule to change to a more helical conformation. In a second case the mutant with the sequence 34AAAAALAAAKAALAAA49, which spans a loop-helix region, had a dramatically altered thermal unfolding transition, suggesting that this region may tend to form a single, uninterrupted, helix. Substitution of Ala38 in the above construct with aspartic acid caused the unfolding to be more like wildtype, suggesting that residue 38, which is at a helix-capping position in the wildtype structure, provides an initiation signal that is essential in the polyalanine mutant for the correct formation of alpha-helix 39-50. In a typical protein, the information that codes for the 3D structure is presumably distributed over many amino acids. The present results suggest that in simplified sequences the key folding information may be restricted to a subset of critical residues, and so be more readily accessible to experimental analysis.

Amino Acid Sequence↗

PubMed2002

A model binding site for testing scoring functions in molecular docking.

Prediction of interaction energies between ligands and their receptors remains a major challenge for structure-based inhibitor discovery. Much effort has been devoted to developing scoring schemes that can successfully rank the affinities of a diverse set of possible ligands to a binding site for which the structure is known. To test these scoring functions, well-characterized experimental systems can be very useful. Here, mutation-created binding sites in T4 lysozyme were used to investigate how the quality of atomic charges and solvation energies affects molecular docking. Atomic charges and solvation energies were calculated for 172,118 molecules in the Available Chemicals Directory using a semi-empirical quantum mechanical approach by the program AMSOL. The database was first screened against the apolar cavity site created by the mutation Leu99Ala (L99A). Compared to the electronegativity-based charges that are widely used, the new charges and desolvation energies improved ranking of known apolar ligands, and better distinguished them from more polar isosteres that are not observed to bind. To investigate whether the new charges had predictive value, the non-polar residue Met102, which forms part of the binding site, was changed to the polar residue glutamine. The structure of the resulting Leu99Ala and Met102Gln double mutant of T4 lysozyme (L99A/M102Q) was determined and the docking calculation was repeated for the new site. Seven representative polar molecules that preferentially docked to the polar versus the apolar binding site were tested experimentally. All seven bind to the polar cavity (L99A/M102Q) but do not detectably bind to the apolar cavity (L99A). Five ligand-bound structures of L99A/M102Q were determined by X-ray crystallography. Docking predictions corresponded to the crystallographic results to within 0.4A RMSD. Improved treatment of partial atomic charges and desolvation energies in database docking appears feasible and leads to better distinction of true ligands. Simple model binding sites, such as L99A and its more polar variants, may find broad use in the development and testing of docking algorithms.

Algorithms↗

PubMed2002

A test of proposed rules for helix capping: implications for protein design.

alpha-helices within proteins are often terminated (capped) by distinctive configurations of the polypeptide chain. Two common arrangements are the Schellman motif and the alternative alpha(L) motif. Rose and coworkers developed stereochemical rules to identify the locations of such motifs in proteins of unknown structure based only on their amino acid sequences. To check the effectiveness of these rules, they made specific predictions regarding the structural and thermodynamic consequences of certain mutations in T4 lysozyme. We have constructed these mutants and show here that they have neither the structure nor the stability that was predicted. The results show the complexity of the protein-folding problem. Comparison of known protein structures may show that a characteristic sequence of amino acids (a sequence motif) corresponds to a conserved structural motif. In any particular protein, however, changes in other parts of the sequence may result in a different conformation. The structure is determined by sequence as a whole, not by parts considered in isolation.

Amino Acid Motifs↗