ABSTRACT

We investigated protein/DNA interactions, using molecular dynamics simulations computed between a 10 Angstrom water layer model of the estrogen receptor (ER) DNA binding domain (DBD) amino acids and DNA of an estrogen response element (ERE) consisting of 25 nucleotide base pairs. This ERE nucleotide sequence occurs naturally upstream of the Xenopus laevis vitellogenin A1 gene. Hydrogen bonding interactions were monitored. In addition, van der Waals and electrostatic interaction energies were calculated. Amino acids of the ER DBD DNA recognition helix formed both direct and water mediated hydrogen bonds at cognate codon- anticodon nucleotide base and backbone sites within the ERE DNA right major groove half-site. These interactions together induced breakage of Watson-Crick nucleotide base pairing hydrogen bonds, resulting in significant structural changes and bending of the DNA into the protein.

INTRODUCTION

The steroid hormone receptor proteins are made up of a family of sequence related DNA regulatory proteins which share common structural modules within their DNA binding domains (DBDs) (1). The DBDs consist of two zinc binding motifs encoded by separate exons (2,3). Adjacent to the first zinc binding motif is a DNA recognition alpha helix (4). Amino acids of this recognition helix specifically interact with nucleotide base sites within the DNA major groove half-sites of the proteins' cognate DNA binding sites. The DNA sites to which the steroid hormone receptor proteins specifically bind are referred to as hormone response elements (HREs) (5). There are differences in nucleotide sequence among the HREs to which the proteins specifically bind (6). Likewise, there are differences in amino acid sequence within the DNA recognition helices of the steroid receptor proteins (4). These differences suggest the existence of a site specific DNA recognition code.

Our laboratory has long been interested in DNA site specific recognition by DNA regulatory proteins and has made several key observations. Earlier, we reported that genetic information is conserved between c-DNA sub-sequences which encode DNA regulatory proteins' DNA binding domains (DBDs) and their cognate sites on DNA to which they specifically bind (4,7,8). As an example, using genetic sequence search techniques, we were the first to locate and describe the Glucocorticoid Receptor (GR) DNA recognition alpha helix on the carboxyl terminus of the first zinc binding motif. We discovered the GR DNA recognition helix by observing that its encoding c-DNA sequence shares genetic information with a glucocorticoid response element (GRE) (4). We compared the genetic sequences of other members of the steroid hormone receptor family to the glucocorticoid receptor sequence. Using this technique, we located putative DNA recognition helices, adjacent to the first zinc binding motif, within the DBDs for other members of the steroid receptor family . This conservation of genetic information prompted us to hypothesize a code for DNA site specific recognition for DNA regulatory proteins. We proposed that DNA site specific recognition is based on stereochemical complementarity between amino acids of the proteins' DBDs and their codon-anticodon nucleotides within cognate DNA binding sites. Recently, we reported atomic interactions from molecular dynamics simulations in solvent between amino acids of the GR DBD in complex with nucleotides within the DNA major groove half-sites of its GRE and flanking regions (9,10). We observed that amino acids of the GR DBD DNA recognition helix interacted with their cognate codon-anticodon nucleotides within the GRE. These findings strongly support our hypothesis for a site specific DNA recognition code as described above.

In the present study, we report that genetic information is conserved between the Estrogen Receptor (ER) DBD (GenBank locus HSERR) and EREs located upstream of the Xenopus laevis vitellogenin A1 gene (GenBank locus XLVITA15). By model building, we observed that amino acids within the ER DBD DNA recognition helix and adjacent beta strand are aligned with their cognate codon-anticodon nucleotides within the ERE DNA major groove half-sites. These findings suggested that amino acids of the ER DBD may interact with their cognate codon-anticodon nucleotides within the ERE DNA major groove half-sites. To investigate this possibility, we conducted 300 picoseconds of molecular dynamics simulations on a model of the ER DBD/ERE complex in a 10 Angstrom water layer. Hydrogen bonding interactions were monitored. In addition, van der Waals and electrostatic interaction energies were calculated. The findings indicate that ER DBD amino acids of the DNA recognition helix and beta strand form both direct and water mediated hydrogen bonds at their cognate codon-anticodon nucleotide base and backbone sites within the ERE DNA major groove half-sites. These interactions together induce breakage of Watson-Crick nucleotide base pairing hydrogen bonds, resulting in significant structural changes and bending of the DNA toward the protein.

MATERIALS AND METHODS

Model Building
The model of the ER DBD dimer used in this study was based on atomic coordinates derived from X-ray crystallography of the ER DBD (11) (PDB structure ID: 1HCQ) A model of B-form DNA of a naturally occurring ERE from (GenBank locus XLVITA15) in which we observed genetic similarity with the ER DBD (12) was likewise created using the NUCLEIC ACID BUILDER module from the QUANTA program (13). Solvated molecular dynamics simulation of the ER DBD/ERE model is described below.

Dynamics Parameters
The solvated molecular dynamics simulations were run on a CRAY YMP C-90 supercomputer using a specially optimized version of CHARMm (release version 22.1) which has an atom limit of 15,000. The 10 Angstrom water layer model required 6717 water atoms, the ER/ERE protein/DNA complex consisted of 2908 atoms resulting in a model of 9625 total atoms. The molecular dynamics simulation required 0.6 CRAY C-90 CPU hours of computational resources per picosecond of simulation.

The solvated model was minimized for 200 cycles using the Steepest Descents method. Then the structure was minimized for 100 cycles using the Adopted Basis Newton-Rapson method. Heating was run for 600 cycles, at 0.001 ps per cycle for a total of 0.6 picoseconds, resulting in 0.5 K temperature increase per cycle (from 0 to 300 degrees K). Equilibration was run for 1000 cycles (1 picosecond) resulting in an overall temperature RMS deviation of approximately 5 degrees K. Finally molecular dynamics were run with a step size of 0.001 picoseconds for an additional 300 picoseconds using velocity scaling. A constant dielectric potential with an E value of 1.00 was used. A non-bonded cutoff of 15.00 angstroms was used. Non-bonded parameters were updated every 20 cycles and all energy terms were computed (see figure 1). For a detailed discussion of the CHARMm potential energy function see reference (14) and for a review of molecular dynamics implementation in the biological sciences see reference (15).

Explicit sodium counter-ions were used in the DNA model, based on geometry provided by Don Gregory Ph.D. from Molecular Simulations Inc. Zinc atoms were placed in the ER structure and tetrahedrally coordinated with the sulfur atoms from the "zinc-finger" cysteines. The residue topology file (RTF) for the "zinc- finger" cysteines was altered and a new residue type was created 'ZCY' (for zinc binding cysteine) in which the negative charges on the sulfur atoms were increased from -0.19 to -0.50 so that the charges from the four tetrahedrally coordinated cysteine sulfur atoms would neutralize the +2.0 charge on the zinc atom. In addition, the charges on the zinc binding cysteine beta carbons were increased from +0.19 to +0.40 and the charges on the alpha carbons were increased from +0.10 to +0.20 in order to maintain the ZCY residue at a net 0.0 charge.

DNA Groove Geometry Calculations
The conformational changes of the DNA during dynamics were evaluated using the CURVES 4.1program provided by Richard Lavery of Laboratoire de Biochimie Theorique CNRS (personal communication). The documentation provided describes CURVES as "an algorithm for calculating a helical parameter description for any irregular nucleic acid segment with respect to an optimal, global helical axis. The solution is obtained by minimizing a function which represents the variations in helical parameters between successive nucleotides as well as quantifying the kinks and dislocations which exist between successive helical axis segments". For more detailed information regarding the CURVES 4.1 program see references (16,17).

Interaction Energy Calculations
Interaction energies were calculated using CHARMm (14,15) between ERE nucleotides and selected ER DBD amino acids from the right monomer of the 10 Angstrom water layer ER DBD/25 base pair ERE model after energy minimization, heating and equilibration (0 picoseconds) and after 300 picoseconds of molecular dynamics . Interaction energy was calculated using a constant dielectric potential with an E value of 1.00. "Total Energy" is the sum of electrostatic interaction energy and Van der Waals interaction energy. The values given for the interaction of particular amino acid and nucleotide residues are the sum of the interaction energies of all atoms in those residues.

Hydrogen Bond Calculations
The hydrogen bond interactions for the 10 Angstrom water layer ER/ERE model were recorded at 1.0 picosecond (1000 cycle) intervals. We used a distance-angle algorithm to compute hydrogen bonds which was based on the results of analysis of hydrogen bonding in proteins (18). The value used for the maximum distance allowed between the hydrogen atom and the acceptor was 2.5 angstroms. The value used for the maximum distance allowed between the atom bearing the hydrogen and the acceptor was 3.3 angstroms. The minimum angle at the acceptor was 90 degrees (limit = 0 to 180 degrees). The minimum angle at the hydrogen was 90 degrees (limit = 0 to 180 degrees). The minimum angle at the atom bearing the hydrogen was 90 degrees (limit = 0 to 180 degrees).

RESULTS AND DISCUSSION

Conservation of Genetic Information
We reported elsewhere that nucleotide sub-sequence similarity exists between well characterized EREs and flanking nucleotides upstream of the Xenopus laevis Vitellogenin A1 gene start site and the c-DNA which encodes amino acids of the ER DBD (12 ). The nucleotide sequence we compared to the ER DBD c-DNA contains two EREs; however, only the ERE site proximal to the Xenopus laevis Vitellogenin A1 gene transcription start site has the "consensus" ERE recognition motif, TGACC, in both DNA major groove half-sites. The distal "non-consensus" ERE contains the sequence TGACT in both major groove half-sites. We observed that primary nucleotide sub-sequence similarity between the ERE and flanking nucleotide sites occurred only with the c-DNA sequence of exon 2 which encodes the first zinc binding motif and DNA recognition helix of the ER DBD (see figure 2a and 2b). We also observed that the c-DNA which encodes the ER DNA recognition helix shares sub-sequence similarity only with the proximal "consensus" ERE site (see figure 2b). However, by examining coding possibilities in all three reading frames on both strands of the "non-consensus ERE", we observed codon sites, embedded in overlapping reading frames, for amino acids of the exon 2 encoded ER DNA recognition helix and exon 3 encoded beta strand (see figure 2c). Furthermore, by model building we observed that amino acids encoded at the splice junctions of exons 2 and 3 of the ER DBD are aligned with their cognate codon-anticodon nucleotides within the "non-consensus" ERE right and left major groove half-sites. This includes amino acids of the ER DNA recognition helix encoded in exon 2 and a beta strand encoded in exon 3 at the splice junction of exons 2/3 (see figure 3a-f).

Molecular Dynamics
The above findings suggested that the amino acids within the ER DBD DNA recognition structures may interact with their cognate codon-anticodon nucleotides within the "non-consensus" ERE and its flanks. To investigate this possibility, we docked the ER DBD dimer at H-bonding distance within the DNA major groove half-sites of the ERE. Using the CHARMm program, we conducted 300 picoseconds of molecular dynamics. An ER DBD/ 25 base pair ERE model, without the water molecules, is shown in figure 4. In this model the ER DBD is docked at approximately 10 Angstroms from the 25 base pair ERE and flanking nucleotides for visual clarity. This model is to be used as a key for locating interactions found between the ER DBD amino acids and nucleotides of the ERE and its flanks during molecular dynamics, see table I.

Amino Acid-Nucleotide Hydrogen Bonding Interactions
Hydrogen bonding interactions between amino acids encoded by exons 2 and 3 of the ER DBD and nucleotides of the ERE were monitored. A summary of H- bonding interactions is shown in table I. Equivalent functional sites on the amino acids are grouped: Lysine hydrogen bond donor sites HZ1, HZ2 and HZ3 are combined as HZ. Arginine hydrogen bond donor sites HH11 and HH12 are combined as HH1 and hydrogen bond donor sites HH21 and HH22 are combined as HH2. Glutamine hydrogen bond donor sites HE21 and HE22 are combined as HE2. Glutamic acid hydrogen bond acceptor sites OE1 and OE2 are combined as OE. Likewise, the DNA backbone phosphate group hydrogen bond acceptor sites O1P and O2P are combined as OP. First and last occurrences of DNA/protein hydrogen bonds which includes minimization, heating and equilibration steps followed by the 300 picosecond production dynamics simulation are given in picoseconds of dynamics with their frequency of occurrence. Frequencies of H-bonding interactions (shown in table I) greater than 300 reflect multiple hydrogen bonds (i.e. when two or more of the grouped atoms from one residue interact at the same atom from another residue) for a given amino acid/nucleotide interaction. The hydrogen bonding interactions between amino acids encoded by exons 2 and 3 of the ER DBD and nucleotides of the ERE were monitored. Individual hydrogen bonds are labeled "C" for amino acid-nucleotide codon interactions, "AC" for amino acid-nucleotide anticodon interactions, "C*" and "AC*" for amino acid-nucleotide codon and anticodon interactions when the codon or anticodon sequence is present reading 3' to 5'. It can be seen in table I that the majority of H-bonding interactions for exon 2 encoded amino acids E203, K206, and K210 of the ER DNA recognition helix occur at codon-anticodon nucleotide sites within the ERE right major groove half-site. Likewise, H-bonding interactions between the ERE right major groove half-site and the exon 3 encoded amino acids K235, S236, and Q238 occur at codon- anticodon nucleotide sites. In contrast, in the left ERE DNA major groove half-site, none of the amino acids of the DNA recognition helix encoded in exon 2 form H- bonds at codon-anticodon sites. However, amino acids K235 and S236 encoded in exon 3 form H-bonds at codon-anticodon nucleotide sites, see table I.

Electrostatic and van der Waals interactions
We calculated van der Waals and electrostatic interaction energies between amino acids of the ER DBD and nucleotides on the sense and antisense strands of the ERE. Calculations were performed on the minimized, heated and equilibrated structures at the beginning of the dynamics simulation and after 300 picoseconds in order to analyze the attractive forces between ER DBD DNA recognition helix amino acids and ERE nucleotides. A total energy (Kcal/M) interaction consisting of both van der Waals and electrostatic energy was determined. Total energy values were recorded for the hydrophilic amino acids of the ER DNA recognition helix and nucleotide base pairs within the ERE DNA right major groove half-site. Glutamic acid 203 has been reported to be important in DNA site specific recognition by the ER protein (11). As can be seen in table I, during the 300 picosecond molecular dynamics Glu 203 forms a direct specific H-bond with the H61 base site of its central codon nucleotide A32, reading 3'- to - 5,' 5'-AAG-3'. However, the energy potential for Glu 203 and nucleotide A32 after 300 picoseconds has an overall positive, repulsive, value, (see figure 5a left). A close-up of Glu 203 interacting with A32 is shown in figure 5a right. A hydrogen bond occurs between Glu 203 sidechain site OE2 and A32 nucleotide base site H61; as expected, a negative, attractive, value is seen between OE2 and H61 sites. An extensive network of water mediated H-bonds also occur between Glu 203 and sites on nucleotides A31, A32 and G33, (see table I and figure 6a). These water mediated interactions together dampen the overall repulsive energy of the base sites of nucleotide A32 and serve to position the Glu 203 sidechain site OE 2 within direct H-bonding distance of the H61 base site of nucleotide A32. Total interactive energy values were also recorded between ERE nucleotides and ER DBD amino acids Lys 206, Lys 210, Gln 214, Lys 235 and Gln 238 sidechains. In all cases, attractive energy values were observed between these amino acids and their cognate codon or anticodon nucleotide base pairs found within the ERE DNA right major groove half-site (see figure 5b-f). In addition, specific H-bonds were formed between the sidechains of Lys 206, Lys 210, Lys 235 and Gln 238 and their cognate codon-anticodon nucleotide sites (see table I and figure 6b,c,e and f).

Specific Amino Acid-Nucleotide Interactions
Genetic information is conserved within the ERE right major groove half-site for amino acids of the exon 2 encoded ER DNA recognition helix Amino acids (see figure 2c and figure 3). In addition, we also observed that genetic information is conserved within both the ERE left and right major groove half-sites for amino acids of the zinc binding motif encoded within exon 3. Amino acids Lys 206, Lys 210 and Arg 211 of the ER DNA recognition helix are conserved at similar positions within the DNA recognition helices of members of the steroid receptor family (4); these amino acids of the ER DNA recognition helix have been reported to specifically bind DNA at ERE sites (11). We observed that amino acids Lys 206, and Lys 210 form both direct and water mediated multidentate H-bonds at cognate codon-anticodon nucleotide base sites within the ERE right major groove half-site, as shown in table I see figure 2c and figure 3a-h for reference. Close up views of specific amino acid-nucleotide H-bonding interactions are shown in figure 6a-f. In figure 6a, a direct H-bond occurs between amino acid Glu 203 and its codon nucleotide A32 (reading 3'-to-5'). Water mediated H-bonds between the sidechain of Glu 203 and its codon nucleotides A31 and G33 (reading 3'-to-5') are also shown. In figure 6b, direct and water mediated H-bonding between Lys 206 and codon nucleotides A32 and G33 respectively are shown. In figure 6c, direct and water mediated H- bonding interactions for amino acid Lys 210 and codon nucleotide G33 are shown. In figure 6d, direct H-bonds are formed between Arg 211 and non- codon nucleotide G16. A water mediated H-bond between Arg 211 and non-codon nucleotide T34 is also shown. In figure 6e, direct H-bonds between exon 3 encoded Lys 235 and its codon nucleotide G39 are shown. Direct and water mediated H- bonds between exon 3 encoded amino acid Gln 238 and its anticodon nucleotides T15 and T14 respectively are shown in figure 6f.

Nucleotide-Nucleotide Hydrogen Bonding Interactions for the 25 bp ERE DNA
Hydrogen bonding interactions between sense and antisense strand ERE nucleotides during 300 picoseconds of molecular dynamics on the ER DBD/ERE 25 bp DNA model are shown in table II. A loss of one or more canonical Watson -Crick (WC) H-bonds can be seen occurring predominantly in the right major groove half-site. This is especially evident at nucleotide base pairs: G16-C35, A17-T34, and C18- G33. The majority of the loss in canonical H-bonds for these nucleotide base pairs can be accounted for by amino acid-nucleotide interactions at WC sites as shown in table I and by the non-canonical nucleotide-nucleotide H-bonding interactions shown in table II. In the left major groove half-site a loss of canonical WC H- bonds occurs between the nucleotide base pair C6-G45 at their H42-06 and N3-H1 base sites. Nucleotide base pair T9-A42 also shows loss of WC H-bonding at the H3-N1 base sites.

Structural Changes in ER DBD Protein/ERE DNA Complex
During molecular dynamics of the ER DBD/ERE complex, structural changes occur in the DNA (see figure 7a-e). The DNA appears to wrap around the ER DBD DNA recognition alpha helices. The minor groove between the left and right ERE major groove half-sites is compressed. The nucleotides in particular within the ERE right major groove half-site show a loss of canonical WC base pairing H-bonds (see table II). A close-up view of the ERE right major groove nucleotide sequence is shown in figure 7d. A loss of WC canonical H-bonding is apparent at nucleotide pairs G16- C35, A17-T34 and C18-G33, see figure 7e. To further illustrate the geometric changes in the ERE DNA, using the CURVES program (16,17), ERE DNA major and minor groove width was analyzed after 300 picoseconds of molecular dynamics for the ER DBD/ERE model compared to ERE DNA at 0 picoseconds (see figure 8). The DNA major and minor groove widths determined at 0 picoseconds, 11.4 and 5.6 Angstroms, respectively are in close agreement with values reported for canonical B-DNA duplexes (17). These values were used to monitor changes in DNA major and minor groove width during molecular dynamics. An increase in width in the ERE right major groove half-site can be seen. A decrease in minor groove width between the ERE DNA major groove half-sites was also observed. These changes in DNA structure reflect loss of nucleotide base pair WC H-bonding which facilitate bending of the DNA into the ER DBD protein. Similar findings of DNA bending have been reported for the ER/ERE and other DNA regulatory protein/DNA complexes (19-21). These conformational changes can be viewed as interactive molecules (see figure 9) and as MPEG movies (see figure 10).

CONCLUSIONS

Our findings, reported herein, show that amino acids Lys 206, Lys 210, and Gln 214 of the ER DNA recognition helix encoded in exon 2 and Lys 235 and Gln 238 of the exon 3 encoded ER DBD second zinc binding motif have electrostatic attraction for their codon-anticodon nucleotides within the ERE right major groove half-site, see figure 5. In addition, these amino acids, with the exception of Gln 214, specifically form both direct and water mediated H-bonds at their cognate codon- anticodon nucleotide base and backbone sites, (see table I and figure 6). Furthermore, Glu 203 forms direct and water mediated H-bonds with its codon nucleotides A31, A32 and G33, reading 3'-to-5', 3'-GAA-5' (see table I and figure 6a). Although an arginine codon-anticodon nucleotide site is not found within the ERE DNA major groove half-sites, the nucleotide trimer TGA does occur. This sequence closely resembles the arginine codon CGA having a T substituted for C thus conserving the pyrimidine at position one of the CGA arginine codon. In addition, the T substitution conserves the stereochemical complementarity between nucleotide base acceptor sites of TGA, as seen with CGA, and the arginine sidechain functional donor sites. Hydrogen bonding interactions between arginine residues of the ER DBD and nucleotides of the ERE occur within the TGA sequence with direct H-bonding interactions occurring predominately at nucleotide base sites on G of the TGA sequence (see table I and figure 6d). Therefore recognition of codon-anticodon nucleotides within the ERE DNA right major groove half-site by amino acids of the ER DNA recognition helix and the exon 3 encoded ER DBD second zinc binding motif offer an explanation for the ER DNA binding preference to ERE major groove half-sites.

Recently, a protein/DNA structure of an ER DBD/ERE complex consisting of an imperfectly palindromic ERE was determined by X-ray crystallography at 2.6A resolution (22). In this study, the right half-site of the ERE contained the "consensus" sequence, 5'-TGACCT-3'- 5'-AGGTCA-3', and the left half-site contained a "non-consensus " sequence, 5'-AAGTCA-3'- 5'-TGACTT-3'. Hydrogen bonding interactions were reported between nucleotides of the "non- consensus" ERE half-site and amino acids of the ER DBD (22). The right half- site of the ERE nucleotide sequence used in our study reported herein is identical to the 5'-AAGTCA-3'- 5'-TGACTT-3' "non-consensus" ERE sequence above (22). Furthermore, the H-bonding interactions reported herein between ER DBD amino acids Tyr 195, His 196, Tyr 197, Glu 203, Gly 204, Lys 210, Lys 211, Arg 211, Arg 234, Lys 235, Gln 238, and Arg 241 and nucleotides of the "non-consensus" ERE sequence (see table I, right column ) are in agreement with the findings reported for the same sequence in the X-ray crystallography structural determination of the ERE/ER DBD complex (22).

Our observations indicate that ER site specific DNA recognition involves overlapping reading frames. In addition, our findings also suggest that site specific DNA recognition may be bi-directional, that is, amino acids may recognize their cognate codon-anticodon nucleotides reading 5'-to-3' or 3'-to-5'. This appears to be the case for Glu 203 of the ER DNA recognition helix as described above. These observations offer an explanation as to why more than one amino acid can interact with the same nucleotide and vise-versa and still satisfy site specific DNA recognition according to our hypothesis. Our findings reported herein and elsewhere indicate that conservation of genetic information (4,7-10,12) is correlated with both DNA site specific recognition and transcription enhancement.

Our findings strongly support the idea of a stereo-chemical basis for the origin of the genetic code (23-32) because amino acids within regulatory proteins' DNA recognition helices are consistently being found lining up with cognate codon-anticodon nucleotides within their specific DNA binding sites (4,7-10,12). These findings also suggest that these structures may have been template dependent in their evolution (i.e. peptides acting as templates for nucleotide polymerization or vice-versa (33-35). The findings of conservation of genetic information between the exon splice junction sites of the ER DBD and its cognate ERE are identical to the findings we reported elsewhere for the glucocorticoid receptor protein and its cognate glucocorticoid response elements (8). Therefore, we propose that prebiotic, template directed autocatalytic synthesis of mutually cognate peptides and polynucleotides resulted in their amplification and evolutionary conservation in contemporary eukaryotic organisms as a modular genetic regulatory apparatus. Finally, the amino acid-nucleotide atomic interactions described herein and elsewhere confirm our original prediction that conservation of genetic information is a determinate of site specific DNA recognition for DNA regulatory proteins (4,7-10,12).

ACKNOWLEDGEMENTS

We thank Don Gregory of Molecular Simulations Inc. for providing geometry for explicit sodium counter- ions used in all simulations and for Zn atom placement and charge parameters for Zn binding cysteines in the "zinc fingers" of the ER DBD structures. We also thank the Molecular Simulations Inc. staff for software support with QUANTA, Michael Fenton of Fentonnet Inc. for data reduction programs, Barry Bolding of Minnesota Supercomputer Center for CHARMm software optimization on the CRAY C-90, Minnesota Supercomputer Institute Scientific Director, Don Truhlar for support and encouragement, the Minnesota Supercomputer Center user services representatives for technical support on the CRAY C-90, R. Lavery for providing CURVES 4.1 software and special thanks are due to Charlie Larson of Silicon Graphics Inc. for hardware support with the IRIS 4D 320-GTX workstation. This work was supported in part by a research grant from the Minnesota Supercomputer Institute, Minneapolis MN. This work was also supported by a research fellowship dedicated to the memory of William Lang Jr..

It is indeed with sadness that we report that our colleague Dr. David F. Hickok passed away on June 5, 1996. This manuscript is dedicated to his memory.

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