A cavity-containing mutant of T4 lysozyme is stabilized by buried benzene

Abstract

The hydrophobic cores of proteins are generally well packed, with few cavities. Mutations in which a bulky buried residue such as leucine or phenylalanine is replaced with a small residue such as alanine can create cavities in the core of a protein (our unpublished results). The sizes and shapes of such cavities can vary substantially depending on factors such as local geometry, whether or not a cavity already exists at the site of substitution, and the degree to which the protein structure relaxes to occupy the space vacated by the substituted residue. We show by crystallographic and thermodynamic analysis that the cavity created by the replacement Leu 99----Ala in T4 lysozyme is large enough to bind benzene and that ligand binding increases the melting temperature of the protein by 6.0 degrees C at pH 3.0. Benzene does not, however, bind to the cavity created by the Phe 153----Ala replacement. The results show that cavities can be engineered in proteins and suggest that such cavities might be tailored to bind specific ligands. The binding of benzene at an internal site 7 A from the molecular surface also illustrates the dynamic nature of proteins, even in crystals.

Capturing Invisible Motions in the Transition from Ground to Rare Excited States of T4 Lysozyme L99A

“…L99A is a model system for studying ligand binding to buried protein cavities and protein excited states, and the conformational changes that govern these phenomena have been enigmatic despite nearly 25 years of experimental study (5,6,(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30). Experimental studies of the L99A mutant of T4 lysozyme have focused on defining the structures of and transition times between the ground state, the excited state, and ligand-bound states.…”

“…Experimental studies of the L99A mutant of T4 lysozyme have focused on defining the structures of and transition times between the ground state, the excited state, and ligand-bound states. Initial crystal structures of the L99A mutant indicate that the~40 Å 3 cavity in the C-terminal domain of wild-type protein expands to an~150 Å 3 cavity (26), a volume capable of accommodating substituted benzenes and noble gases (20,24,(27)(28)(29)(30). O 2 is simulated to bind and escape the buried cavity through openings between the D, E, G, H, and J helices (31), whereas binding of substituted benzenes is accompanied by discrete rearrangements in the F and G helices (30).…”

RosettaHoles: Rapid assessment of protein core packing for structure prediction, refinement, design, and validation

“…Protein cores are packed as tightly as corresponding crystals, and mutations that disrupt a protein core strongly reduce the free energy of folding. [1][2][3] The near absence of voids in protein cores is in part a reflection of the large free energy cost of forming a protein-sized cavity in water, which increases steeply with the total volume of the structure, including voids.…”

Structure-Based Engineering of Internal Cavities in Coiled-Coil Peptides,

“…enzyme specificities, creating novel binding sites, and studying protein stability (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12), but despite the diverse roles of natural cavities in biology, high-resolution structural investigations of model cavities in the aqueous milieu are rare. We believe studying designed cavities within synthetic peptides may provide models for understanding natural binding sites as well as designing novel receptors or biocatalysts.…”