Kevin and Jana Huerter allowed me to rejoice in the joy of their children Alex, Justin. I shudder to think how different my life would be were it not for the education they fought to provide and the values they instilled through their examples.
Overview of the Thesis
The higher generation of β-alanine dendrimers contain internal depressions connected by channels that run along the entire polymer unit. The third section of the thesis focuses on the results of our studies of dihydrofolate reductase (DHFR) complexes.
GENERATION
The crystal structure of the E.coli DHFR/MTX binary complex (9) shows that the inhibitor binds in a kinked manner in the active site (Figure 8). The orientations of the MTX and DHF pteridine rings in the active site of D HFR.
34; DHF NH
NADPH
Polyamidoamine Starburst Dendrimers
CORE
MONOMER
The synthetic scheme used in the production of these dendrimers falls into the category of redundant reagents. The first step in the synthesis involves the extensive Michael addition of methyl acrylate to an appropriate amine group, either the core unit or the terminal amino groups of the previous generation. The resulting esters are then exhaustively amidated with excess ethylenediamine to produce the next full generation of polyamidoamine.
Termination of this synthetic cascade after condensation of methyl acrylate produces ester-terminated oligomers, referred to as half-generation materials. 34; This is the number of atoms actually included in the simulation; i.e., it ignores the hydrogen atoms attached to the carbon since they are handled implicitly.
CORE MONOMER
Molecular Simulation Details
- End-to-end Distances
- Radius of Gyration
- Moments of Inertia
- Surface Calculations
- Molecular Volumes
The solvent-accessible surface is created by rolling a sphere with the radius of the solvent (1.4 A for water) around the vdw surface. Thus, the accessible area produced continues to increase as the radius of the probe sphere increases. Representations of 8-alanine starbursts of the early generation of starfish-like dendrimers (one to four).
Tile views of molecular surface vdw screens for early generation starburst 8-alanine dendrimers (one to four) illustrating the absence of an internal region of these polymers. Tile views of vdw molecular surface screens for the next generation of 8-alanine starburst dendrimers (five to seven) illustrating the extensive internal channels and cavities associated with these materials.
End-to-end Distances
The addition of the vdw molecular surface, as depicted in Figure 13, clearly shows the close-packed properties of the interior of these materials. Wireframe representations of the penta-erythritol, polyether star-burst dendrimers (generations one through four). Flattened views of the vdw molecular surface are shown for the penta-erythritol polyether starburst dendrimers (generations one to four) illustrating the crowding and lack of extensive, internal, void spaces associated with these materials.
Due to differences in the length and flexibility of the penta-erythritol units relative to the ,8-alanine fragment, these polymers range from ∼13 Å to ∼40 Å (generations 1 to 4) with an average increase at each generation of ∼9A. This difference is most likely due to the fact that the simulated structures mitigate some of the jamming by allowing some slight distortions in the bond and angular components of the system.
Radius of Gyration
These plots show that the early generation of ,8-alanines show a linear increase in Rg as a function of monomer number, indicating the open, extended conformation adopted by these molecules. For the polyether series, the change in R1 as a function of the total number of monomer units gradually increases exponentially, indicating a spherical shape. To quantify the shape changes of the macromolecules with formation, the total moments of inertia and their components along the principal axes are listed in Table 7.
For the β-alanine series, the asymmetry of the structures clearly decreases as the number of monomers in the polymer increases. These components of the moment of inertia indicate that above generation 4 the P AMAM polymers have the shape of an oblate spheroid.
Surface Calculations
- Normalized Accessible Surface Area
The area of the accessible surfaces, calculated at different probe radii for the ,8-alanine and polyether dendrimers, is reported in Tables 8 and 9. In fact, this deviation from linearity at small p makes it possible to determine what fraction of the calculated accessible surface. due to internal depressions. For the polyether polymers, no more than ∼20% of the available surface area is associated with internal regions of the molecule.
At generation 7, however, we find that the inner surface is 120% of the outer surface. For generation 5 and 6, the internal surface is 30% and 70% of the amount of external surface area, respectively.
Volume Calculations
- Total Accessible Surface Volumes
- Normalized Accessible Surface Volumes
Available surface volume data (presented in Tables 18 and 19) also allow calculation of the volume change per added monomer. This suggests that the accessible surfaces generated for the polyether dendrimers fit Eq. Ln (volume) versus Ln (area) at different probe radius values for polyether starburst dendrimers.
Ln (vol.) vs. Ln (area.), at different values of the probe radius, for PAMAM starburst dendrimers. Its size and shape are suitable for the cavities and channels found in the higher generation B-alanine systems.
DENDRIMER CATECHOL
These inner regions have an area approximately equal to that found on the outside of the polymer spheroid. The inclusion of Chicken DHFR yields only ~60% chemical homology to the bacterial forms, suggesting subtle differences in the character of the active site. The solvent surface of the active site is mapped by the array of dots.
Asp-27 (dark blue), involved in protonation of the substrate pteridine ring, is shown salt-bridged to the MTX. Phe-31 (green) and Leu-54 (blue) are strictly conserved residues that form part of the substrate binding site.
HCHO
Crystal Structures
The conformation of the bound methotrexate inhibitor is also quite similar in the two crystals. A strictly conserved Phe ring (31 Ee, 30 Le) is involved in nonpolar contacts with both the pterin and benzoyl rings of the bound inhibitor. From the crystal study of the ternary complex, the only contacts found between the adenine ring of NADPH and the enzyme are hydrophobic.
The binding of nicotinamide ribose appears to be quite specific, making hydrophobic contacts with a number of invariant residues (Ile-3, Gly-14 and Gly-99 Le) and H-bonding through its hydroxyl groups to its carbonyl oxygen. -18 (Le) and the side chains of Asp-125 and Ser-48. The nicotinamide functional group of NADPH resides in a pocket created by largely invariant amino acid residues.
Isotope Studies
The overall fold of the Chicken form agrees quite well with that of the bacterial forms, as illustrated in Figure 4. The major differences between the bacterial and Chicken tertiary structures involve inserted amino acid residues that are typically restricted to loop regions on the outside of the protein. A schematic overview of the orientation of the nicotinamide (NADPH) and pteridine (DHF) reaction centers involved in hydride transfer.
Spectroscopy reveals that the isotope is transferred to the surface of C7 in folate. The enzyme-catalyzed hydride transfer, for the reduction of both DHF and folate, involves the same side of NADPH and the same side of the folic acid substrate.
Kinetic Profile
During the enzymatic reduction of folic acid, the 4-pro·R hydrogen is again transferred to the pteridine ring. The off-rate for THF from the DHFR • THF • NADP+ ternary complex is 2.4 sec-1, while the loss of NADP+ reduces this rate to 1.4 sec-1. The addition of NADPH to the enzyme complex significantly increases the product off rate to 12.5 sec-1.
Thus, the release of THF is facilitated by the presence of the reduced NADPH cofactor, suggesting another role for the NADPH cofactor. These values indicate that changes at position 54 (a strictly conserved leucine residue) to Ile or Gly lead to the following changes in kinetics: a) substrate binding affinity decreases by more than one order of magnitude; (b) the rate of hydride transfer is reduced by more than 30 times; and (c) the steady-state release rate of the product increases more than eightfold.
The Active Site Asp-27
- Coordinate Transformation
- Identification of the Site Regions
NADPH active sites were identified in an analogous manner, but based only on the position of L. For these two DHFR complexes, a slight discrepancy in the definition of the NADPH active site was encountered. The binding criterion is clearly illustrated by the agreement in the structure of the two bound MTX molecules (red from E. coli and blue from L. caaei).
For the NADPH site, 22 of the 59 residues are the same, leading to a sequence homology of 37%. 2) ,ide: where the side chains align with the bound substrate and/or cofactor, but without reasonable correspondence between the backbone sites;.
Chicken DHFR
A comparison of the site sequences for the three forms of DHFR analyzed in this study is provided in Table 4. For the substrate binding site only 15 of the 52 possible residues (29%) are equivalent, while in the NADPH binding site only 14 of the 59 positions (24%) are sequence homologous. Thus, for the entire region of the catalytic site, there is only 24% sequence homology, i.e. 20 of the possible 85 residues.
Thus, we should expect that the free energy profile for the steady-state kinetic pathway of chicken DHFR will not be as close as the two bacterial systems. The specifics of the approaches used to determine the initial structures for the simulations are presented below.
Models for Starting Complexes
- Substrate and Inhibitor Orientation
The water molecules along the empty NADPH site were removed from the starting ternary form and appropriate counterions were positioned near the anionic sites of the NADPH. The other changes involved the position of the protein loop that held the NADPH in its binding site. Therefore, the orientation of this fragment in the E.coli protein was modeled after that of the L.
On the other hand, ca+2 was located at the periphery of the protein structure. MTX and eleven regions of the protein were defined and used for segmented solvation.
S Site Mutations
After the above manipulations, the final binary complex simulation solvent consisted of 619 waters (197 experimental and 422 modeled into the system), 23 Na+ (all modeled waters converted to ions) and 13 c1- (1 experimental and 12 modeled waters converted to ions ). For the ternary complexes with the natural substrate DHF, 21 leads were deleted from the NADPH binding site. The initial structure for the solvent in the active site region was examined using solvent accessible surfaces.21•22 Solvent accessible molecular surfaces for the bound inhibitor or substrate, the NADPH cofactor, the active site of the protein and the water molecule within 8 Å of the resulting bound small molecules (with a probe radius of 1 ,4 A).
Thus, an appropriate amount of solvent was present in the active zone. cont) Glutamate Residues HOH Number Distance0. The coordinates of the atoms in this new loop were assigned from those of the docked Chicken structure file.
Force Field
For the electrostatic energies, the partial charges for the amino acid residues were the values of AMBER. The atomic charges for methotrexate, dihydrofolate, folate and NADPH were based on values reported for analogous amino and nucleic acid fragments, and are given in Table 6.
Simulation Protocols
- Energy Minimization and Molecular Dynamics
