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...

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The internal dynamics of native and immobilized Escherichia coli dihydrofolate reductase (DHFR) have been examined using incoherent quasielastic neutron scattering. These results reveal no difference between the high frequency vibration mean-square displacement of the native and the immobilized E. coli DHFR. However, length-scale-dependent, picosecond dynamical changes are found. On longer length scales, the dynamics are comparable for both DHFR samples. On shorter length scales, the dynamics is dominated by local jump motions over potential barriers. The residence time for the protons to stay in a potential well is tau = 7.95 +/- 1.02 ps for the native DHFR and tau = 20.36 +/- 1.80 ps for the immobilized DHFR. The average height of the potential barrier to the local motions is increased in the immobilized DHFR, and may increase the activation energy for the activity reaction, decreasing the rate as observed experimentally. These results suggest that the local motions on the picosecond timescale may act as a lubricant for those associated with DHFR activity occurring on a slower millisecond timescale. Experiments indicate a significantly slower catalytic reaction rate for the immobilized E. coli DHFR. However, the immobilization of the DHFR is on the exterior of the enzyme and essentially distal to the active site, thus this phenomenon has broad implications for the action of drugs distal to the active site.

Bovine testicular hyaluronidase (BTH) has been used as a spreading factor for many years and was primarily characterized by its enzymatic activity. As recombinant human hyaluronidases are now available the bovine preparations can be replaced by the human enzymes. However, data on the pH-dependent activity of hyaluronidases reported in literature are inconsistent in part or even contradictory. Detection of the pH-dependent activity of PH-20 type hyaluronidases, i.e. recombinant human PH-20 (rhPH-20) and BTH, showed a shift of the pH optimum from acidic pH values in a colorimetric activity assay to higher pH values in a turbidimetric activity assay. Contrarily, recombinant human Hyal-1 (rhHyal-1) and bee venom hyaluronidase (BVH) exhibited nearly identical pH profiles in both commonly used types of activity assays. Analysis of the hyaluronic acid (HA) degradation products by capillary zone electrophoresis showed that hyaluronan was catabolized by rhHyal-1 continuously into HA oligosaccharides. BTH and, to a less extent, rhPH-20 exhibited a different mode of action: at acidic pH (pH 4.5) HA was degraded as described for rhHyal-1, while at elevated pH (pH 5.5) small oligosaccharides were produced in addition to HA fragments of medium molecular weight, thus explaining the pH-dependent discrepancies in the activity assays. Our results suggest a sub-classification of mammalian-type hyaluronidases into a PH-20/BTH and a Hyal-1/BVH subtype. As the biological effects of HA fragments are reported to depend on the size of the molecules it can be speculated that different pH values at the site of hyaluronan degradation may result in different biological responses.