Conformational characteristics of 4-acetylcytidine found in tRNA

The conformational characteristics of a modified nucleoside 4-acetylcytidine (ac4C) was analyzed by proton nuclear magnetic resonance spectroscopy. This nucleoside was found to take the C3'-endo form predominantly. The enthalpy difference between the C2'-endo form and the C3'-endo form of the ribose moiety of ac4C was determined and 4-acetylation was found to stabilize the C3'-endo form further by 0.83 kcal.mol-1. This conformational characteristics of ac4C is probably important for the correct codon recognition and the stability of the tertiary structure of tRNA molecule.     

Influence of pH on the equilibrium association constants for oligodeoxyribonucleotide-directed triple helix formation at single DNA sites.

The energetics of oligodeoxyribonucleotide-directed triple helix formation for the pyrimidine.purine.pyrimidine structural motif were determined over the pH range 5.8-7.6 at 22 degrees C (100 mM Na+ and 1 mM spermine) using quantitative affinity cleavage titration. The equilibrium binding constants for 5'-TTTTTCTCTCTCTCT-3' (1) and 5'-TTTTTm5CTm5CTm5CTm5CTm5CT-3' (2, m5C is 2'-deoxy-5-methylcytidine) increased by 10- and 20-fold, respectively, from pH 7.6 to 5.8, indicating that the corresponding triple-helical complexes are stabilized by 1.4 and 1.7 kcal.mol-1, respectively, at the lower pH. Replacement of the five cytosine residues in 1 with 5-methylcytosine residues to yield 2 affords a stabilization of the triple helix by 0.1-0.4 kcal.mol-1 over the pH range 5.8-7.6. An analysis of these data in terms of a quantitative model for a general pH-dependent equilibrium transition revealed that pyrimidine oligonucleotides with cytidine and 5-methylcytidine form local triple-helical structures with apparent pKa's of 5.5 (C+GC triplets) and 5.7 (m5C+GC triplets), respectively, and that the oligonucleotides should bind to single sites on large DNA with apparent affinity constants of approximately 10(6) M-1 even above neutral pH.     

The structure of 3'-O-anthraniloyladenosine, an analogue of the 3'-end of aminoacyl-tRNA.

3'-O-Anthraniloyladenosine, an analogue of the 3'- terminal aminoacyladenosine residue in aminoacyl-tRNAs, was prepared by chemical synthesis, and its crystal structure was determined. The sugar pucker of 3'-O-anthraniloyladenosine is 2'-endo resulting in a 3'-axial position of the anthraniloyl residue. The nucleoside is insynconformation, which is stabilized by alternating stacking of adenine and benzoyl residues of the neighboring molecules in the crystal lattice. The conformation of the 5'-hydroxymethylene in 3'-O- anthraniloyladenosine is gauche-gauche. There are two intramolecular and two intermolecular hydrogen bonds and several H-bridges with surrounding water molecules. The predominant structure of 3'-O-anthraniloyladenosine in solution, as determined by NMR spectroscopy, is 2'-endo,gauche-gauche and anti for the sugar ring pucker, the torsion angle around the C4'-C5'bond and the torsion angle around the C1'-N9 bond, respectively. The 2'-endo conformation of the ribose in 2'(3')-O-aminoacyladenosine, which places the adenine and aminoacyl residues in equatorial and axial positions, respectively, could serve as a structural element that is recognized by enzymes that interact with aminoacyl-tRNA or by ribosomes to differentiate between aminoacylated and non-aminoacylated tRNA.     

Histidine residues at the N- and C-termini of alpha-helices: perturbed pKas and protein stability.

A single histidine residue has been placed at either the N-terminus or the C-terminus of each of the two alpha-helices of barnase. The pKa of that histidine residue in each of the four mutants has been determined by 1H NMR. The pKas of the two residues at the C-terminus are, on average, 0.5 unit higher, and those of the residues at the N-terminus are 0.8 unit lower, than the pKa of histidines in unfolded barnase at low ionic strength. The conformational stability of the mutant proteins at different values of pH has been measured by urea denaturation. C-Terminal histidine mutants are approximately 0.6 kcal mol-1 more stable when the introduced histidine is protonated, both at low and high ionic strength. N-Terminal mutants with a protonated histidine residue are approximately 1.1 kcal mol-1 less stable at low ionic strength and 0.5 kcal mol-1 less stable at high ionic strength (1 M NaCl). The low-field 1H NMR spectra of the mutant proteins at low pH suggest that the C-terminal histidines form hydrogen bonds with the protein while the N-terminal histidines do not form the same. The perturbations of pKa and stability result from a combination of different electrostatic environments and hydrogen-bonding patterns at either ends of helices. The value of 0.6 kcal mol-1 represents a lower limit to the favorable electrostatic interaction between the alpha-helix dipole and a protonated histidine residue at the C-terminal end of the helix.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA triplex structures are stabilized by the incorporation of 3'-endo blocked pyrimidine nucleosides in the Hoogsteen strand

A short route to pyrimidine locked nucleosides has been developed for their incorporation in triplex forming oligonucleotides (TFO). Compared to oligonucleotides built with standard nucleosides, the modified TFOs containing 3'-endo blocked residues formed, with their corresponding DNA duplexes, more stable triple helix systems, an effect which might be ascribed to the 3'-endo pucker of the modified nucleoside residues.     

Wobble modification differences and subcellular localization of tRNAs in Leishmania tarentolae: implication for tRNA sorting mechanism

In Leishmania tarentolae, all mitochondrial tRNAs are encoded in the nuclear genome and imported from the cytosol. It is known that tRNA(Glu)(UUC) and tRNA(Gln)(UUG) are localized in both cytosol and mitochondria. We investigated structural differences between affinity-isolated cytosolic (cy) and mitochondrial (mt) tRNAs for glutamate and glutamine by mass spectrometry. A unique modification difference in both tRNAs was identified at the anticodon wobble position: cy tRNAs have 5-methoxycarbonylmethyl-2- thiouridine (mcm(5)s(2)U), whereas mt tRNAs have 5- methoxycarbonylmethyl-2'-O-methyluridine (mcm(5)Um). In addition, a trace portion (4%) of cy tRNAs was found to have 5-methoxycarbonylmethyluridine (mcm(5)U) at its wobble position, which could represent a common modification intermediate for both modified uridines in cy and mt tRNAs. We also isolated a trace amount of mitochondria-specific tRNA(Lys)(UUU) from the cytosol and found mcm(5)U at its wobble position, while its mitochondrial counterpart has mcm(5)Um. Mt tRNA(Lys) and in vitro transcribed tRNA(Glu) were imported much more efficiently into isolated mitochondria than the native cy tRNA(Glu) in an in vitro importation experiment, indicating that cytosol-specific 2-thiolation could play an inhibitory role in tRNA import into mitochondria.     

Structure-activity relationships in engineered proteins: characterization of disruptive deletions in the alpha-ammonium group binding site of tyrosyl-tRNA synthetase

Residues Asp-78 and Gln-173 of the tyrosyl-tRNA synthetase of Bacillus stearothermophilus form part of the binding site for tyrosine by making hydrogen bonds with the alpha-ammonium group. Asp-38 is close enough to the group to make an important electrostatic contribution. Unlike other residues in the active site that have been studied by site-directed mutagenesis, Asp-38, Asp-78, and Gln-173 are part of hydrogen-bonded networks. Each of these residues has been mutated to an alanine, and the resultant mutants have been studied by kinetics to construct the difference energy diagrams for the formation of tyrosyl adenylate. In each example, the binding of tyrosine is weakened by about 2.5 kcal mol-1. But, unlike previous mutants, the dissociation of the second substrate, in this case ATP, is also seriously affected, being weakened by some 2 kcal mol-1 for TyrTS(Ala-78) and TyrTS(Ala-173). The energy of the transition state for the formation of tyrosyl adenylate is raised by 7.8 kcal mol-1 for the former and 4.5 kcal mol-1 for the latter mutant. Addition of these mutants to linear free energy plots constructed for the nondisruptive mutants in the accompanying study [Fersht, A. R., Leatherbarrow, R. J., & Wells, T. N. C. (1987) Biochemistry (preceding paper in this issue)] reveals large deviations of the data for TyrTS(Ala-38) and TyrTS(Ala-78) from the regression line. These thus belong to a different class of mutations from previous nondisruptive examples. This observation combined with the structural evidence and difference energy diagrams strongly suggests that the mutations Asp----Ala-38 and Asp----Ala-78 are disruptive in nature.     

CNDO/2 molecular orbital calculations on the antifolate DAMP and some related species: structural geometries, ring distortions, change distributions and conformational characteristics

Geometry-optimized CNDO/2 molecular orbital calculations were carried out on 2, 4-diamino-5-(1-adamantyl 1)-6-methyl pyrimidine (DAMP), a potent inhibitor of mammalian dihydrofolate reductase which is now in clinical trials, and on its inactive 5-(1-naphthyl) analogue (DNMP-1). Crystallographic data show that DAMP (as the ethylsulfonate salt) has a severely distorted, N1 protonated, pyrimidine ring and has steric crowding of the 6-methyl and adamantyl hydrogens whereas DNMP-2 (as a methanol complex) has a planar, nonprotonated pyrimidine ring that is nearly perpendicular to the naphthalene ring. The CNDO/2 results largely reproduce the crystal structure geometry and show that the ring distortions in DAMP are initiated by steric conflicts between the adamantyl group and the 4- and 6-substituents on the ring. In DNMP-1, the non-interfering naphthyl ring induces little strain within the pyrimidine ring and the effect of protonation is negligible. Rotation about the bond joining the two ring groups is restricted in DAMP by a broad barrier of ca. 8.0 kcal mol-1, and no conformation was successful in relieving steric conflicts and hence reducing the ring distortions. In DNMP-1, rotation is less hindered overall with a broad region of accessible conformational space and a maximum barrier of ca. 7.2 kcal mol-1 for the coplanar conformation. The electronic charge distributions of DAMP and DNMP-1 are almost identical and protonation is preferred at N1 rather than at N3 by ca. 3.7 kcal mol-1 for both DAMP and DNMP-1. The calculations establish that the present methodology can be useful as a predictive tool with regard to the structure and conformational characteristics of these and related species.     

Conformational properties of branched RNA fragments in aqueous solution

The conformational properties of branched trinucleoside diphosphates ACC, ACG, AGC, AGG, AUU, AGU, AUG, ATT, GUU, and aAUU [XYZ = X(2'p5'Y)3'p5'Z] have been studied in aqueous solution by nuclear magnetic resonance (1H, 13C), ultraviolet absorption, and circular dichroism. It is concluded from these studies that the purine ring of the central residue (X; e.g., adenosine) forms a base-base stack exclusively with the purine or pyrimidine ring of the 2'-nucleotidyl unit (Y; 2'-residue). The residue attached to the central nucleoside via the 3'-5'-linkage (Z; 3'-residue) is "free" from the influence of the other two heterocyclic rings. The ribose rings of the central nucleoside and the 2'- and 3'-residues exist as equilibrium mixtures of C2'-endo (2E)-C3'-endo (3E) conformers. The furanose ring of the central nucleoside (e.g., A) when linked to a pyrimidine nucleoside via the 2'-5'-linkage shows a higher preference for the 2E pucker conformation (e.g., AUG, AUU, ACG, ca. 80%) than those linked to a guanosine nucleoside through the same type of bond (AGU, AGG, AGC, ca. 70%). This indicates some correlation between nucleotide sequence and ribose conformational equilibrium. The 2E-3E equilibrium of 2'-pyrimidines (Y) shows significant, sometimes exclusive, preference (70-100%) for the 3E conformation; 3'-pyrimidines and 2'-guanosines have nearly equal 2E and 3E rotamer populations; and the ribose conformational equilibrium of 3'-guanosines shows a preference (60-65%) for the 2E pucker. Conformational properties were quantitatively evaluated for most of the bonds (C4'-C5', C5'-O5', C2'-O2', and C3'-O3') in the branched "trinucleotides" AUU and AGG by analysis of 1H-1H, 1H-31P, and 13C-31P coupling constants. The C4'-C5' bond of the adenosine units shows a significant preference for the gamma + conformation. The dominant conformation about C4'-C5' and C5'-O5' for the 2'-and 3'-nucleotidyl units is gamma + and beta t, respectively, with larger gamma + and beta t rotamer populations for the 2'-unit. The increased conformational purity in the 2'-residue, compared to the 3'-residue, is ascribed to the presence of an ordered (adenine----2'-residue) stacked state. The favored rotamers about C3'-O3' and C2'-O2' are epsilon- and epsilon'-, respectively. The conformational features of AUU and AGG were compared to those of their constitutive dimers A3'p5'G, A2'p5'G, A3'p5'U, and A2'p5'U and monomers 5'pG and 5'pU.     

Selective restoration of the selenoprotein population in a mouse hepatocyte selenoproteinless background with different mutant selenocysteine tRNAs lacking Um34

Novel mouse models were developed in which the hepatic selenoprotein population was targeted for removal by disrupting the selenocysteine (Sec) tRNA([Ser]Sec) gene (trsp), and selenoprotein expression was then restored by introducing wild type or mutant trsp transgenes. The selenoprotein population was partially replaced in liver with mutant transgenes encoding mutations at either position 34 (34T-->A) or 37 (37A-->G) in tRNA([Ser]Sec). The A34 transgene product lacked the highly modified 5-methoxycarbonylmethyl-2'-O-methyluridine, and its mutant base A was converted to I34. The G37 transgene product lacked the highly modified N(6)-isopentenyladenosine. Both mutant tRNAs lacked the 2'-methylribose at position 34 (Um34), and both supported expression of housekeeping selenoproteins (e.g. thioredoxin reductase 1) in liver but not stress-related proteins (e.g. glutathione peroxidase 1). Thus, Um34 is responsible for synthesis of a select group of selenoproteins rather than the entire selenoprotein population. The ICA anticodon in the A34 mutant tRNA decoded Cys codons, UGU and UGC, as well as the Sec codon, UGA. However, metabolic labeling of A34 transgenic mice with (75)Se revealed that selenoproteins incorporated the label from the A34 mutant tRNA, whereas other proteins did not. These results suggest that the A34 mutant tRNA did not randomly insert Sec in place of Cys, but specifically targeted selected selenoproteins. High copy numbers of A34 transgene, but not G37 transgene, were not tolerated in the absence of wild type trsp, further suggesting insertion of Sec in place of Cys in selenoproteins.     

Determination of the activation energy for pseudorotation of the furanose ring in nucleosides by 13-C nuclear-magnetic-resonance relaxation.

The activation energies for the pseudorotation of the furanose ring in adenosine, guanosine, inosine and xanthosine dissolved in liquid deuteroammonia have been determined by analysis of the longitudinal relaxation rates of the single tertiary carbons between +40 degrees C and minus 60 degrees C. For the purine ribosides the average activation energy was found to be 4.7 plus or minus 0.5 kcal x mol-1 (20 plus or minus 2 kJ x mol-1). For the pyrimidine nucleosides cytidine and uridine the respective activation energy should be higher since it could not be determined by 13-C relaxation measurements. This result can be explained by the formation of a hydrogen bond between the 5'-hydroxymethyl group and the base. In adenosine, guanosine, inosine and xanthosine the relaxation rates of C(5') are smaller than all others thus excluding the formation of a hydrogen bond between the purine base and the 5'-hydroxymethyl group of a strength comparable to the one suggested for cytidine and uridine.     

The folding of an enzyme. II. Substructure of barnase and the contribution of different interactions to protein stability.

Barnase is described anatomically in terms of its substructures and their mode of packing. The surface area of hydrophobic residues buried on formation and packing of the structural elements has been calculated. Changes in stability have been measured for 64 mutations, 41 constructed in this study, strategically located over the protein. The purpose is to provide: (1) information on the magnitudes of changes in stabilization energy for mutations of residues that are important in maintaining the structure; and (2) probes for the folding pathway to be used in subsequent studies. The majority of mutations delete functional moieties of side-chains or make isosteric changes. The energetics of the interactions are variable and context-dependent. The following general conclusions may be drawn, however, from this study about the classes of interactions that stabilize the protein. (1) Truncation of buried hydrophobic side-chains has, in general, the greatest effect on stability. For fully buried residues, this averages at 1.5 kcal mol-1 per methylene group with a standard deviation of +/- 0.6 kcal mol-1. Truncation of partly exposed leucine, isoleucine or valine residues that are in the range of 50 to 80 A2 of solvent-accessible area (30 to 50% of the total solvent-accessible area on a Gly-X-Gly tripeptide, i.e. those packed against the surface) has a smaller, but relatively constant effect on stability, at 0.81 kcal mol-1 per methylene group with a statistical standard deviation of +/- 0.18 kcal mol-1. (2) There is a very poor correlation between hydrophobic surface area buried and the free energy change for an extensive data set of hydrophobic mutants. The best correlation is found to be between the free energy change and the number of methylene groups within a 6 A radius of the hydrophobic groups deleted. (3) Burial of the hydroxyl group of threonine in a pocket that is intended for a gamma-methyl group of valine costs 2.5 kcal mol-1, in the range expected for the loss of two hydrogen bonds.(ABSTRACT TRUNCATED AT 400 WORDS)     

Crystal structure of d(GCGCGCG) with 5''-overhang G residues.

The crystal structure of the DNA heptamer d(GCGCGCG) has been solved at 1.65 A resolution by the molecular replacement method and refined to an R-value of 0.184 for 3598 reflections. The heptamer forms a Z-DNA d(CGCGCG)2 with 5'-overhang G residues instead of an A-DNA d(GCGCGC)2 with 3'-overhang G residues. The overhang G residues from parallel strands of two adjacent duplexes form a trans reverse Hoogsteen G x G basepair that stacks on the six Z-DNA basepairs to produce a pseudocontinuous helix. The reverse Hoogsteen G x G basepair is unusual in that the displacement of one G base relative to the other allows them to participate in a bifurcated (G1)N2 . . . N7(G8) and an enhanced (G8)C8-H . . . O6(G1) hydrogen bond, in addition to the two usual hydrogen bonds. The 5'-overhang G residues are anti and C2'-endo while the 3'-terminal G residues are syn and C2'-endo. The conformations of both G residues are different from the syn/C3'-endo for the guanosine in a standard Z-DNA. The two cobalt hexammine ions bind to the phosphate groups in both GpC and CpG steps in Z(I) and Z(II) conformations. The water structure motif is similar to the other Z-DNA structures.     

Nucleotide sequence of nuclear 5.4 S RNA of mouse cells

The nucleotide sequence of nuclear 5.4 S RNA, a new species of small nuclear RNA (snRNA) of mouse cells, was determined. The 5.4 S RNA consists of 138 nucleotide residues containing 1 mol each of 2,2,7- trimethylguanosine (m3(2,2,7) G), 2'-O-methyladenosine (Am), 2'-O-methyluridine (Um) and pseudouridine as modified nucleosides. This RNA has a cap structure, m3(2,2,7) ++GpppAm -, at its 5'-terminus and sequences complementary to the terminal consensus sequences of introns. The sequence complementary to the 5'-splice junction, A-U-C-C-psi-U-A-C-C-U-G, is very similar to the 5'-terminal sequence of U1 RNA.     

tRNA1Ser(G34) with the anticodon GGA can recognize not only UCC and UCU codons but also UCA and UCG codons

The tRNA1Ser (anticodon VGA, V=uridin-5-oxyacetic acid) is essential for translation of the UCA codon in Escherichia coli. Here, we studied the translational abilities of serine tRNA derivatives, which have different bases from wild type at the first positions of their anticodons, using synthetic mRNAs containing the UCN (N=A, G, C, or U) codon. The tRNA1Ser(G34) having the anticodon GGA was able to read not only UCC and UCU codons but also UCA and UCG codons. This means that the formation of G-A or G-G pair allowed at the wobble position and these base pairs are noncanonical. The translational efficiency of the tRNA1Ser(G34) for UCA or UCG codon depends on the 2'-O-methylation of the C32 (Cm). The 2'-O-methylation of C32 may give rise to the space necessary for G-A or G-G base pair formation between the first position of anticodon and the third position of codon.     

Solution structure of RNA duplexes containing alternating CG base pairs: NMR study of r(CGCGCG)2 and 2'-O-Me(CGCGCG)2 under low salt conditions.

Structures of r(CGCGCG)2 and 2'-O-Me(CGCGCG)2 have been determined by NMR spectroscopy under low salt conditions. All protons and phosphorus nuclei resonances have been assigned. Signals of H5'/5" have been assigned stereospecifically. All 3JH,H and 3JP,H coupling constants have been measured. The structures were determined and refined using an iterative relaxation matrix procedure (IRMA) and the restrained MD simulation. Both duplexes form half-turn, right-handed helices with several conformational features which deviate significantly from a canonical A-RNA structure. Duplexes are characterised as having C3'-endo sugar pucker, very low base-pair rise and high helical twist and inclination angles. Helices are overwound with <10 bp per turn. There is limited inter-strand guanine stacking for CG steps. Within CG steps of both duplexes, the planes of the inter-strand cytosines are not parallel while guanines are almost parallel. For the GC steps this pattern is reversed. The 2'-O-methyl groups are spatially close to the 5'-hydrogens of neighbouring residues from the 3'-side and are directed towards the minor groove of 2'-O-Me(CGCGCG)2 forming a hydrophobic layer. Solution structures of both duplexes are similar; the effect of 2'-O-methylation on the parent RNA structure is small. This suggests that intrinsic properties imposed by alternating CG base pairs govern the overall conformation of both duplexes.     

Desulfurization of 2-thiouracil nucleosides: conformational studies of 4-pyrimidinone nucleosides

4-Pyrimidinone ribofuranoside (H(2)o(4)U) and 4-pyrimidinone 2'-deoxyribofuranoside (dH(2)o(4)U) were synthesized by the oxidative desulfurization of parent 2-thiouracil nucleosides with m-chloroperbenzoic acid. The crystal structures of H(2)o(4)U and dH(2)o(4)U and their conformations in solution were determined and compared with corresponding 2-thiouracil and uracil nucleosides. The absence of a large 2-thiocarbonyl/2-carbonyl group in the nucleobase moiety results in C2'-endo puckering of the ribofuranose ring (S conformer) in the crystal structure of H(2)o(4)U, which is not typical of RNA nucleosides. Interestingly, the hydrogen bonding network in the crystals of dH(2)o(4)U stabilizes the sugar moiety conformation in the C3'-endo form (N conformer), rarely found in DNA nucleosides. In aqueous solution, dH(2)o(4)U reveals a similar population of the C2'-endo conformation (65%) to that of 2'-deoxy-2-thiouridine (62%), while the 62% population of the S conformer for H(2)o(4)U is significantly different from that of the parent 2-thiouridine, for which the N conformer is dominant (71%). Such a difference may be of biological importance, as the desulfurization process of natural tRNA 2-thiouridines may occur under conditions of oxidative stress in the cell and may influence the decoding process.     

Co-operative interactions during protein folding.

The theory for measuring co-operativity between interactions in proteins by protein engineering experiments is developed by introducing a procedure for analysing increasing orders of synergy in a protein with increasing numbers of residues. The (pairwise) interaction energy (delta 2Gint) between two side-chains may be measured experimentally by a double-mutant cycle consisting of the wild-type protein, the two single mutants and the double mutant. This procedure may be extended to three residues to give a value for delta 3Gint for a triple-mutant cube, and to higher orders using multi-dimensional mutant space. We now show that delta 3Gint is the excess energy of adding all three chains compared with the sum of all the pairwise values of delta 2Gint for each of the constituent double-mutant cycles and the sum of all the single addition energies. This physical interpretation extends to higher orders of mutation. delta nGint (i.e. the interaction energy for n residues), thus, reveals the layers of synergy in interactions as a protein is built up. This procedure is applied to measuring changes in synergy during the refolding of barnase for the triad of salt-linked residues Asp8, Asp12 and Arg110, which are mutated to alanine residues. The value of delta 3Gint in the folded structure is 0.77(+/- 0.06) kcal mol-1 (i.e. the triad is 0.77 kcal mol-1 more stable than expected from the sum of the individual pairwise interactions and single contributions). The value of delta 3Gint is still significant in the transition state for unfolding (0.60(+/- 0.07) kcal mol-1) and in the folding intermediate (0.60(+/- 0.13 kcal mol-1)). These results show that synergistic interactions exist in barnase, in its transition state for unfolding and in a refolding intermediate. A direct measurement of the change of co-operativity between the folded state and the transition state for unfolding shows a decrease of 0.17(+/- 0.04) kcal mol-1, suggesting that the initial stages of protein unfolding may be accompanied by some loosening of structure in parts that still interact. The similar extent of co-operativity in the transition state for unfolding and the intermediate in refolding suggests that the intermediate is homogeneous, at least in the region of the salt-linked triad, as heterogeneity would lower the co-operativity.     

Environmentally induced conformational changes in B-type DNA: comparison of the conformation of the oligonucleotide d(TCGCGAATTCGCG) in solution and in its crystalline complex with the restriction nuclease EcoRI

Raman spectroscopic analysis of the secondary structure of the crystalline restriction endonuclease EcoRI, the oligonucleotide d(TCGCGAATTCGCG) in solution, and the corresponding crystalline EcoRI-oligonucleotide complex reveals structural differences between the complexed and uncomplexed protein and oligonucleotide components that appear to be linked to complex formation. Structural differences that are spectroscopically identified include (1) an increase in the population of furanose rings adopting the C3'-endo conformation and (2) spectroscopically observed changes in base stacking which are probably associated with the crystallographically observed distortion of the phosphate backbone about positions C(3)-G(4) and C(9)-G(10) and unwinding between the symmetry-related segments GAA-TTC which make up the central recognition core (McClarin et al., 1986). Changes in base stacking due to distortions and unwinding along the oligonucleotide result in differences in the base vibrational region between the spectra of the complex and the oligonucleotide in solution. The spectroscopic analysis indicates that the C2'-endo population is similar for the oligonucleotide in solution and in the complex. The additional C3'-endo population in the complex appears to arise from the conversion of rings adopting alternative conformations such as C1'-exo and O1'-endo. Analysis of the vibrational bands derived from guanine indicates that the population of guanine residues associated with furanose rings in a C2'-endo conformation is similar for the oligonucleotide in solution and in the crystalline complex. This implies that the increase in C3'-endo population is not associated with guanine residues. Large conformational distortions such as those observed in the crystal distortions are not observed in either the crystal or the solution of the oligomer d(CGCGAATTCGCG).(ABSTRACT TRUNCATED AT 250 WORDS)