Lessons from the lysozyme of phage T4

Baase, W.A.; Liu, Lijun; Tronrud, Dale E.; Matthews, Brian W.

doi:10.1002/pro.344

Cited by 192 publications

(240 citation statements)

References 65 publications

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“…The crystal forms obtained were all different from those observed before for T4L. 55 The molecular structures and crystal packing diagrams are shown in Figures 2 and 3, and crystallographic statistics are shown in Supporting Information Table 1.…”

Section: T4l Histidine Mutantsmentioning

confidence: 67%

An approach to crystallizing proteins by metal‐mediated synthetic symmetrization

Laganowsky

Zhao²,

Soriaga

et al. 2011

Protein Science

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Combining the concepts of synthetic symmetrization with the approach of engineering metal-binding sites, we have developed a new crystallization methodology termed metal-mediated synthetic symmetrization. In this method, pairs of histidine or cysteine mutations are introduced on the surface of target proteins, generating crystal lattice contacts or oligomeric assemblies upon coordination with metal. Metal-mediated synthetic symmetrization greatly expands the packing and oligomeric assembly possibilities of target proteins, thereby increasing the chances of growing diffraction-quality crystals. To demonstrate this method, we designed various T4 lysozyme (T4L) and maltose-binding protein (MBP) mutants and cocrystallized them with one of three metal ions: copper (Cu 21 ), nickel (Ni 21 ), or zinc (Zn 21 ). The approach resulted in 16 new crystal structures-eight for T4L and eight for MBP-displaying a variety of oligomeric assemblies and packing modes, representing in total 13 new and distinct crystal forms for these proteins. We discuss the potential utility of the method for crystallizing target proteins of unknown structure by engineering in pairs of histidine or cysteine residues. As an alternate strategy, we propose that the varied crystallization-prone forms of T4L or MBP engineered in this work could be used as crystallization chaperones, by fusing them genetically to target proteins of interest.

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Section: T4l Histidine Mutantsmentioning

confidence: 67%

An approach to crystallizing proteins by metal‐mediated synthetic symmetrization

Laganowsky

Zhao²,

Soriaga

et al. 2011

Protein Science

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“…T4 lysozyme (T4L) was selected for study because of the large database of crystal structures of ligand-binding cavity mutations and the corresponding thermodynamic characterization from Matthews and coworkers (4,(12)(13)(14)(15). Remarkably, comparison of the WT and mutant structures showed very little difference or limited local relaxation near the cavity, although the mutations were strongly destabilizing (4, 15).…”

mentioning

confidence: 99%

Conformational selection and adaptation to ligand binding in T4 lysozyme cavity mutants

López

Yang

Altenbach

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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The studies presented here explore the relationship between protein packing and molecular flexibility using ligand-binding cavity mutants of T4 lysozyme. Although previously reported crystal structures of the mutants investigated show single conformations that are similar to the WT protein, site-directed spin labeling in solution reveals additional conformational substates in equilibrium exchange with a WT-like population. Remarkably, binding of ligands, including the general anesthetic halothane shifts the population to the WT-like state, consistent with a conformational selection model of ligand binding, but structural adaptation to the ligand is also apparent in one mutant. Distance mapping with double electron-electron resonance spectroscopy and the absence of ligand binding suggest that the new substates induced by the cavity-creating mutations represent alternate packing modes in which the protein fills or partially fills the cavity with side chains, including the spin label in one case; external ligands compete with the side chains for the cavity space, stabilizing the WT conformation. The results have implications for mechanisms of anesthesia, the response of proteins to hydrostatic pressure, and protein engineering.EPR | site-directed spin labeling | DEER | benzene | saturation recovery G lobular proteins have overall packing densities similar to those of crystalline solids (1) but nevertheless contain cavities and pockets that can range from a few to hundreds of cubic angstroms (2, 3). These native packing defects are generally destabilizing (4, 5), and improving the packing of the protein interior may be a general way to increase protein stability. Indeed, small-to-large mutations that fill native cavities can increase stability (6-8), but with a concomitant loss of function (7,8). Thus, cavities in the protein interior can play a critical role in function. One role of cavities may be to allow alternative packing arrangements of the core. The resulting ensemble of conformational substates could give rise to promiscuity in protein-protein interactions (9) and allosteric behavior (7,8,10). In addition, large-to-small mutations that generate cavities may have played an important role in the evolution of protein function (11).Considering the potentially important role of cavities in sculpting the energy landscape of proteins, the present study was undertaken to investigate, in solution, the structural and dynamical response of a protein to engineered cavities introduced by large-tosmall mutations. T4 lysozyme (T4L) was selected for study because of the large database of crystal structures of ligand-binding cavity mutations and the corresponding thermodynamic characterization from Matthews and coworkers (4,(12)(13)(14)(15). Remarkably, comparison of the WT and mutant structures showed very little difference or limited local relaxation near the cavity, although the mutations were strongly destabilizing (4, 15).In the present study, we examined the cavity-creating mutants L121A/L133A, L133G, and W138A in sol...

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“…This near equivalence explains why the formation of a p-helix is not a universal occurrence and that, at least in lysozyme, maintenance of alpha helicity appears to be more common. 23,24 The fact that the p-helical conformation is favored in villin headpiece despite being destabilized to a greater degree than the energetic cost of exposing the interface between leucine 61 and the hydrophobic pocket to water (3.5 vs. 3.3 kcal/mol) suggests that extending the alpha helix would displace helix 5 and disrupt the overall topology of villin headpiece (similar to the deletion mutant, HP67 D61).…”

Section: Discussionmentioning

confidence: 99%

On the unyielding hydrophobic core of villin headpiece

2012

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Villin headpiece (HP67) is a small, autonomously-folding domain that has become a model system for understanding the fundamental tenets governing protein folding. In this communication, we explore the role that Leu61 plays in the structure and stability of the construct. Deletion of Leu61 results in a completely unfolded protein that cannot be expressed in Escherichia coli. Omission of only the aliphatic leucine side chain (HP67 L61G) perturbed neither the backbone conformation nor the orientation of local hydrophobic side chains. As a result, a large, solventexposed hydrophobic pocket, a negative replica of the leucine side-chain, was created on the surface. The loss of the hydrophobic interface between leucine 61 and the hydrophobic pocket destabilized the construct by~3.3 kcal/mol. Insertion of a single glycine residue immediately before Leu61 (HP67 L61[GL]) was also highly destabilizing and had the effect of altering the backbone conformation (a-helix to p-helix) in order to precisely preserve the wild-type position and conformation of all hydrophobic residues, including Leu61. In addition to demonstrating that the hydrophobic side-chain of Leu61 is critically important for the stability of villin headpiece, our results are consistent with the notion that the precise interactions present within the hydrophobic core, rather than the hydrogen bonds that define the secondary structure, specify a protein's fold.

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Lessons from the lysozyme of phage T4

Cited by 192 publications

References 65 publications

An approach to crystallizing proteins by metal‐mediated synthetic symmetrization

An approach to crystallizing proteins by metal‐mediated synthetic symmetrization

Conformational selection and adaptation to ligand binding in T4 lysozyme cavity mutants

On the unyielding hydrophobic core of villin headpiece

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