Jeffrey G. Mandell's research while affiliated with University of California, San Diego and other places

The computer program DOT quickly finds low-energy docked structures for two proteins by performing a systematic search over six degrees of freedom. A novel feature of DOT is its energy function, which is the sum of both a Poisson-Boltzmann electrostatic energy and a van der Waals energy, each represented as a grid-based correlation function. DOT evaluates the energy of interaction for many orientations of the moving molecule and maintains separate lists scored by either the electrostatic energy, the van der Waals energy or the composite sum of both. The free energy is obtained by summing the Boltzmann factor over all rotations at each grid point. Three important findings are presented. First, for a wide variety of protein-protein interactions, the composite-energy function is shown to produce larger clusters of correct answers than found by scoring with either van der Waals energy (geometric fit) or electrostatic energy alone. Second, free-energy clusters are demonstrated to be indicators of binding sites. Third, the contributions of electrostatic and attractive van der Waals energies to the total energy term appropriately reflect the nature of the various types of protein-protein interactions studied.

Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry was used to identify peptic fragments from protein complexes that retained deuterium under hydrogen exchange conditions due to decreased solvent accessibility at the interface of the complex. Short deuteration times allowed preferential labeling of rapidly exchanging surface amides so that primarily solvent accessibility changes and not conformational changes were detected. A single mass spectrum of the peptic digest mixture was analyzed to determine the deuterium content of all proteolytic fragments of the protein. The protein-protein interface was reliably indicated by those peptides that retained more deuterons in the complex compared with control experiments in which only one protein was present. The method was used to identify the kinase inhibitor [PKI(5-24)] and ATP-binding sites in the cyclic-AMP-dependent protein kinase. Three overlapping peptides identified the ATP-binding site, three overlapping peptides identified the glycine-rich loop, and two peptides identified the PKI(5-24)-binding site. A complex of unknown structure also was analyzed, human alpha-thrombin bound to an 83-aa fragment of human thrombomodulin [TMEGF(4-5)]. Five peptides from thrombin showed significantly decreased solvent accessibility in the complex. Three peptides identified the anion-binding exosite I, confirming ligand competition experiments. Two peptides identified a new region of thrombin near the active site providing a potential mechanism of how thrombomodulin alters thrombin substrate specificity.

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) was used to determine amide proton/deuteron (H/D) exchange rates. The method has broad application to the study of protein conformation and folding and to the study of protein-ligand interactions and requires no modifications of the instrument. Amide protons were allowed to exchange with deuterons in buffered D2O at room temperature, pD 7.25. Exchanged deuterons were "frozen" in the exchanged state by quenching at pH 2.5, 0 degree C and analyzed by MALDI-TOF MS. The matrix mixture consisted of 5 mg/mL alpha-cyano-4-hydroxycinnamic acid, acetonitrile, ethanol, and 0.1% TFA. The matrix was adjusted to pH 2.5, and the chilled MALDI target was rapidly dried. Deuteration of amide protons on cyclic AMP-dependent protein kinase was measured after short times of incubation in deuterium by pepsin protein digestion and MALDI-TOF MS analysis. The unseparated peptic digest was analyzed in a single spectrum of the mixture. From five spectra, H/D exchange rates were determined for some 40 peptides covering 65% of the protein sequence.

Molecular biologists, biochemists, and biophysicists are often faced with the problem of trying to determine how two molecules might interact. This information is necessary for a fundamental understanding of the mechanisms of biology, but can be very difficult to extract. Experimental methods are widely available for determining whether or not two molecules interact, but these generally depend on thermodynamic or kinetic measurements which do not shed great light on the nature and location of the interacting portions of the molecules in question. Structural studies by crystallography or NMR can be definitive, but are frequently difficult, costly, and time-consuming. Techniques of molecular biology such as deletion analysis and site directed mutagenesis are powerful, but laborious unless a starting point can be found. Completely theoretical methods are generally even slower than experimental methods and have significant uncertainty unless carefully cross-checked against experimental data. However, theoretical models can give great insight into the nature of the interactions. This insight is arguably the most valuable product of theoretical analysis of interacting systems.

The purpose of the molecular interaction program DOT (Daughter of Turnip) is rapid computation of the electrostatic potential energy between two proteins or other charged molecules. DOT exhaustively tests all six degrees of freedom, rotational and translational, and produces a grid of approximate interaction energies and orientations. It is able to do this because the problem is cast as the convolution of the potential field of the first molecule and any rotated charge distribution of the second. The algorithm lends itself to both parallelization and vectorization, permitting huge increases in computational speed over other methods for obtaining the same information. For example, a complete mapping of interactions between plastocyanin and cytochrome c was done in eight minutes using 256 nodes of an Intel Paragon. DOT is expected to be particularly useful as a rapid screen to find configurations for more detailed study using exact energy models.