Direct Determination of Vibrational Density of States Change on Ligand Binding to a Protein

Abstract

The change in the vibrational density of states of a protein (dihydrofolate reductase) on binding a ligand (methotrexate) is determined using inelastic neutron scattering. The vibrations of the complex soften significantly relative to the unbound protein. The resulting free-energy change, which is directly determined by the density of states change, is found to contribute significantly to the binding equilibrium. DOI: 10.1103/PhysRevLett.93.028103 PACS numbers: 87.15.He An understanding of how ligands bind to proteins is of fundamental importance in biology and medicine [1][2][3][4][5][6]. Protein:ligand association has been assumed to be dominated by factors such as the hydrophobic effect, hydrogen bonding, electrostatic, and van der Waals interactions. However, as early as 1963 it was suggested that an additional mechanism might exist, due to increased flexibility in the protein:ligand complex manifested by a change in the spectrum of vibrations due to formation in the complex of new, intermolecular interactions [7][8][9][10][11][12][13]. Theoretical normal mode analyses, used to estimate this vibrational change on insulin dimerization [12] and on water binding to bovine pancreatic trypsin inhibitor [13], have suggested that the effect is likely to be thermodynamically important. However, experimental determination of the vibrational change has been lacking.Inelastic neutron scattering, in which the dynamic structure factor Sq; ! is measured as a function of the scattering wave vector q and energy transfer h! (where ! is the angular frequency), has been used to determine the vibrational density of states (frequency distribution) g! for several proteins [14 -16]. Here we present an experimental determination of the change in g! on binding a ligand to a protein. This determination allows thermodynamic quantities associated with the vibrational change to be derived. The enzyme chosen is dihydrofolate reductase (DHFR), an important target for anticancer and antibacterial drugs [17][18][19][20][21]. DHFR catalyzes the reduction of dihydrofolate to tetrahydrofolate in the presence of the nicotinamide adenine dinucleotide phosphate (NADPH) cofactor. The ligand used is methotrexate (MTX), a folate antagonist of DHFR that has been used effectively as a cytotoxic agent in the treatment of cancers [22].To minimize scattering from solvent molecules, the system was exchanged with D 2 O. To do this, lyophilized DHFR from E.coli was dissolved in D 2 O equilibrated at 4 C overnight and freeze dried. NADPH and NADPH MTX were added in equimolar ratios to the enzyme. As the dissociation constants of DHFR with NADPH and MTX are low (K For the neutron experiments, the samples were contained in an aluminum sample holder. Sample amounts were 98.1 mg (uncomplexed) and 108.6 mg (complexed). The measurements were performed on the time-of-flight spectrometer IN6 at the Institut Laue-Langevin (ILL), Grenoble, with an incident neutron beam wavelength of 5.12 Å . The scattering experiments were performed at 120 K to ensure that all dyn...

Substrate adhesion of a nongrafted flexible polymer in a cavity

“…This also includes numerous detailed studies, e.g., of polymer film wetting phenomena [6,7], pattern recognition [8,9], protein-ligand binding and docking [10,11,12], charged adsorbed polymers [13] as well as deposition and growth of polymers at surfaces [14].…”

Anharmonic transitions in nearly dry l-cysteine I

“…The discussed possibilities include quantum effects near a zero point vibration [7], and methyl group rotation associated with anharmonicity [2,8]. Vibrational modes play a very important role in the conformational arrangement of different functional states and also in the energetic balance of the chemical bonds [9]. Theoretical studies on the an- harmonic effects on phonons show that their frequency and linewidth analyzed by the inelastic light scattering (Raman effect) could vary due to the anharmonic potential shape of the simple oscillator and also due to phononphonon interactions [10,11].…”

Pickin’ Up Good Vibrations

“…Indeed, dating back to the 1980s in a series of calculations on different systems, this effect was shown to favor binding (5-7), and this was finally confirmed by density of states measurements on the binding of methotrexate to dihydrofolate reductase using inelastic neutron scattering (8).…”