Response of a Protein Structure to Cavity-Creating Mutations and Its Relation to the Hydrophobic Effect

Abstract

Six "cavity-creating" mutants, Leu46----Ala (L46A), L99A, L118A, L121A, L133A, and Phe153----Ala (F153A), were constructed within the hydrophobic core of phage T4 lysozyme. The substitutions decreased the stability of the protein at pH 3.0 by different amounts, ranging from 2.7 kilocalories per mole (kcal mol-1) for L46A and L121A to 5.0 kcal mol-1 for L99A. The double mutant L99A/F153A was also constructed and decreased in stability by 8.3 kcal mol-1. The x-ray structures of all of the variants were determined at high resolution. In every case, removal of the wild-type side chain allowed some of the surrounding atoms to move toward the vacated space but a cavity always remained, which ranged in volume from 24 cubic angstroms (A3) for L46A to 150 A3 for L99A. No solvent molecules were observed in any of these cavities. The destabilization of the mutant Leu----Ala proteins relative to wild type can be approximated by a constant term (approximately 2.0 kcal mol-1) plus a term that increases in proportion to the size of the cavity. The constant term is approximately equal to the transfer free energy of leucine relative to alanine as determined from partitioning between aqueous and organic solvents. The energy term that increases with the size of the cavity can be expressed either in terms of the cavity volume (24 to 33 cal mol-1 A-3) or in terms of the cavity surface area (20 cal mol-1 A-2). The results suggest how to reconcile a number of conflicting reports concerning the strength of the hydrophobic effect in proteins.

Functional analyses and molecular modeling of two c-Kit mutations responsible for imatinib secondary resistance in GIST patients

“…The V to A substitution removes two carbon atoms; the calculated DDG bind for V654A mutant disfavors the mutant by 1.52 kcal/mol, resulting in a destabilization of approximately 0.8 kcal/mol per carbon atom upon cavity formation. This estimate is in good agreement with the results of earlier experimental studies (Kellis et al, 1988;Eriksson et al, 1992;Pace et al, 1996;Loladze et al, 2002) and theoretical calculations (Nicholls et al, 1991;Lee, 1993;Lazaridis et al, 1995). Replacing V with A at position 654 further reflects only in a slight alteration of some other imatinib contact points (E640, T670, C673 and F811) and some ATP-binding residues (K623 and E671).…”

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

“…Remarkably, there is a high degree of cavity plasticity, exceeding what might be expected from previous crystallographic studies (18,19), that involves residues, metastates, and conformations beyond the discrete crystallographic states observed for L99A bound to a congeneric series of benzene-related compounds (24,30). Further, the number of rotamers observed for buried L99A residues exceeds that seen in the several-hundred-microsecond simulations of other protein cores, such as ubiquitin, RNaseH, and b-lactamase (56).…”

“…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.…”

Empirical free energy calculation: Comparison to calorimetric data

“…Second, a proteinprotein interface is not always well packed. In fact, unfavorable vdW interactions or cavities reduce binding, but do not affect the free energy function if vdW cancellation is assumed (Eriksson et al, 1992;Vajda et al, 1994).…”