Goutam Gupta

Untenability of the Heteronomous DNA Model for Poly(dA) · Poly(dT) in Solution. This DNA Adopts a Right-Handed B-DNA Duplex in Which the Two Strands are Conformationally Equivalent. A 500 MHz NMR Study Using One Dimensional NOE

1D NOE 1H NMR spectroscopy at 500 MHz was employed to examine the structure of poly(dA).poly(dT) in solution. NOE experiments were conducted as a function of presaturation pulse length (50, 30, 20 and 10 msec) and power (19 and 20 db) to distinguish the primary NOEs from spin diffusion. The 10 msec NOE experiments took 49 hrs and over 55,000 scans for each case and the difference spectra were almost free from diffusion. The spin diffused NOE difference spectra as well as difference NOE spectra in 90% H2O + 10% D2O in which TNH3 was presaturated enabled to make a complete assignment of the base and sugar protons. It is shown that poly(dA).poly(dT) melts in a fashion in which single stranded bubbles are formed with increasing temperature.

Poly(dA-dT)•Poly(dA-dT) in Low Salt Appears to be a Left-Handed B-Helix Combined Use of Chemical Theory, Fiber Diffraction and NMR Spectroscopy

Poly(dA-dT).poly(dA-dT) can adopt the B- and D- forms in the fibrous state. Theoretical energy calculations and fiber diffraction analyses suggest that there can be three structural models of poly(dA-dT).poly(dA-dT) in each of these two forms viz right and left-handed Watson Crick models and left-handed Hoogsteen--a total of six possible models. Fiber data for the polymer in the B- or the D-form or energy calculations cannot distinguish any one model from the other. However, a comparison of observed proton chemical shifts with the theoretically computed ones and the NOE studies on exchangeable and nonexchangeable protons suggest that poly(dA-dT).poly(dA-dT) in low salt solution exists predominantly in the left-handed B-conformation.

NOE data at 500 MHz reveal the proximity of phenyl and tyrosine rings in enkephalin

Met5‐enkephalin ‐ a pentapeptide (Tyr‐Gly‐Gly‐Phe‐Met) ‐ can exist in two possible folded arrangements with a rigid two‐hydrogen‐bonded network. In one arrangement, a Gly 2‐Gly 3 β‐bend is formed and in the other a Gly 3‐Phe 4 β‐bend. The two conformations are distinguished by the spatial relation of Tyr 1 and Phe 4: in the Gly 2‐Gly 3 β‐bend, Tyr 1 and Phe 4 can be brought close to each other while in the Gly 3‐Phe 4 β‐bend they are far apart (⪢5 Å). We have utilized one‐dimensional (1D) nuclear Overhauser effect (NOE) measurements between the ring protons of Tyr 1 and Phe 4 to determine their proximity. The NOE data clearly show that a pair protons, one each from Tyr 1 and Phe 4, are as close as 3.3 Å while other inter‐proton distances are beyond 4.5 Å. Therefore, we propose the presence of a Gly 2‐Gly 3 β‐bend (in which Tyr 1 and Phe 4 are spatially close) for Met5‐enkephalin in solution. The structure of Met5‐enkephalin in solution is very similar to the single crystal structure of Leu5‐enkephalin and tends to explain the biological activity data of several modified enkephalins.

Solution Structure of Poly(dA-dT)· Poly(dA-dT) in Low and High Salt: A 500 MHz 1H NMR Study Using One-Dimensional NOE

CD spectra of poly(dA-dT).poly(dA-dT) in low salt (10-100 mM NaCl) and high salt (4-6 M CsF) are different i.e. 275 nm band gets inverted in going from low to high salt (Vorhickova et. al., J. Mol. Biol. 166, 85, 1983). However, from CD spectra alone it is not possible to decipher any structural differences that might exist between the low and high salt forms of poly(dA-dT).poly(dA-dT). Hence, we took recourse to high resolution NMR spectroscopy to understand the structural properties of poly(dA-dT).poly(dA-dT) in low and high salt. A detailed analysis of shielding constants and extensive use of NOE studies under minimum spin diffusion conditions using C(8)-deuterated poly(dA-dT).poly(dA-dT) enabled us to come up with the following conclusions (i) base-pairing is Watson-Crick under low and high salt conditions. (ii) under both the conditions of salt the experimental data can be explained in terms of an equilibrium blend of right and left-handed B-DNA duplexes with the left-handed form 70% and the right-handed 30%. In a 400 base pairs long poly(dA-dT).poly(dA-dT) (as used in this study), equilibrium between right and left-handed helices can also mean the existence of both helical domains in the same molecule with fast interchange between these domains or/and unhindered motion/propagation of these domains along the helix axis. (iii) However, there are other structural differences between the low and high salt forms of poly(dA-dT).poly(dA-dT); under the low salt condition, right- and left-handed B-DNA duplexes have mononucleotide as a structural repeat while under the high salt conditions, right- and left-handed B-DNA duplexes have dinucleotide as a structural repeat. In the text we provide the listing of torsion angles for the low and high salt structural forms. (iv) Salt (CsF) induced structural transition in poly(dA-dT).poly(dA-dT) occurs without any breakage of Watson-Crick pairing. (v) The high salt form of poly(dA-dT).poly(dA-dT) is not the left-handed Z-helix. Although the results above from NMR data are quite unambiguous, a question still remains i.e. what does the salt (CsF) induced change in the CD spectra of poly(dA-dT).poly(dA-dT) really indicate? Interestingly, we could show that the salt (CsF) induced change in poly(dA-dT).poly(dA-dT) is quite similar to that caused by a basic polypeptide viz. poly-L(Lys2-Ala)n i.e. both the agents induced a psi-structure in DNA.

A cytosine·cytosine base paired parallel DNA double helix with thymine·thymine bulges

500 MHz 1H NMR studies using 2D‐NOESY indicate that the oligonucleotide d(CTCTCT) at low pH forms a parallel double helix with cytosine·cytosine base pairs and thymine·thymine bulges. This unusual structure may explain the hypersensitivity of S1 nuclease at low pH towards supercoiled plasmids containing d(CT) n inserts.

Structure and Dynamics of Netropsin-Poly(dA-dT)· Poly(dA-dT) Complex: 500 MHz 1H NMR Studies

Antibiotic netropsin is known to bind specifically to A and T regions in DNA; the mode of binding being non-intercalative. Obviously, H-bonding between the proton donors of netropsin and acceptors N3 of A and O2 of T comes as a strong possibility which might render this specificity. In netropsin there could be 8 proton donors: four terminal amino groups and four internal imino groups. However, methylation of the terminal amino groups does not alter the binding affinity of netropsin to DNA--but the modification of the internal imino groups significantly lowers the binding affinity. Hence, the logical conclusion is that netropsin may specifically interact with A and T through H-bonding and in order to do so, it should approach the helix from the minor groove. The present paper provides experimental data which verify the conclusion mentioned above. Using poly(dA-dT).poly(dA-dT) as a model system it was observed following a thorough theoretical stereochemical analysis that netropsin could bind to -(T-A-T) sequence of the polymer in the B-form through the minor groove by forming specific H-bonding. Models could be either right or left-handed B-DNA with a mono or dinucleotide repeat. By monitoring the 31P signals of free poly(dA-dT).poly(dA-dT) and netropsin-poly(dA-dT).poly(dA-dT) complex we show that the drug changes the DNA structure from essentially a mononucleotide repeat to that of very dominant dinucleotide repeat; however the base-pairing in the DNA-drug complex remain to be Watson-Crick. Whether H-bonding is the specific mode of interaction was judged by monitoring the imino protons of netropsin in the presence of poly(dA-dT).poly(dA-dT). This experiment was conducted in 90% H2O + 10% D2O using the time-shared long pulse. It was found that exchangeable imino protons of netropsin appear in the drug-DNA complex and disappear upon increasing the D2O content; thus confirming that H-bonding is indeed the specific mode of interaction. From these and several NOE measurements, we propose a structure for poly(dA-dT).poly(dA-dT)-netropsin complex. In summary, experimental data indicate that netropsin binds to poly(dA-dT).poly(dA-dT) by forming specific hydrogen bonds and that the binding interaction causes the structure to adopt a Watson-Crick paired dinucleotide repeat motif.(ABSTRACT TRUNCATED AT 400 WORDS)

Netropsin Specifically Recognizes One of the Two Conformationally Equivalent Strands of Poly (dA)·Poly (dT). One Dimensional NMR Study at 500 MHz Involving NOE Transfer Between Netropsin and DNA Protons

Recent observations that the heteronomous structural model for poly(dA).poly(dT) is not found in solution and that in this DNA, the two strands are conformationally equivalent (J. Biomole. Str. Dyns. 2, 1057 (1985], has added a new dimension to the structural dynamics of DNA-netropsin complex. Does the antibiotic somehow distinguish between the two strands and specifically interact with only one of the conformationally equivalent strands? Model-building studies suggest that netropsin can either bind to the dA-strand in the minor groove such that H-bonds are formed between the imino protons N4-H, N6-H, N8-H of netropsin and N3 atoms of A or can bind to the dT-strand in the minor groove and form H-bonds between the imino-protons N4-H, N6-H, N8-H of netropsin and O2 atoms of T. If netropsin binds to the dA-strand, AH2 atoms of poly(dA).poly(dT) would be in closer proximity to the imino protons N4-H, N6-H, N8-H and pyrrole ring protons C5-H, C11-H of netropsin than they would be, if netropsin binds to the dT-strand. In order to distinguish these possibilities experiments were conducted which involved NOE energy transfer between netropsin and DNA protons in the drug-DNA complex. Difference NOE spectra of netropsin-poly(dA).poly(dT) complex in which AH2 was irradiated indicate that dominant NOEs were observed at the imino and pyrrole ring protons of netropsin. When the netropsin pyrrole ring protons were irradiated, the magnetization transfer was at AH2 of DNA. These observations suggest that netropsin binds to the dA-strand of poly(dA).poly(dT) even though dA/dT strands are conformationally equivalent.

During B-Z Transition There is No Large Scale Breakage of Watson-Crick Base Pairs A Direct Demonstration Using 500 MHz 1H NMR Spectroscopy

Monitoring of the Watson-Crick GNH1 proton in poly(dG-dC).poly(dG-dC) at 500 MHz in 90% H2O:10% D2O at 30 degrees C as a function of NaCl concentration (1.5 to 3.6 M), demonstrates that the bases retain Watson-Crick pairing throughout the transition. This observation unequivocally demonstrates that during the B-Z transition there is no large scale and detectable base pair opening and that macroscopically the phenomenon can be described as a direct helix to helix transition. We present frame by frame, an energetically sound stereodynamical trajectory for this transfiguration from right-handed B-DNA to left-handed Z-DNA.

Structure of poly(dA).poly(dT) is not identical to the AT rich regions of the single crystal structure of CGCGAATTBrCGCG. The consequence of this to netropsin binding to poly(dA).poly(dT).

The basic assumption of Dickerson and Kopka (J. Biomole. Str. Dyns. 2, 423, 1985) that the conformation of poly(dA).poly(dT) in solution is identical to the AT rich region of the single crystal structure of the Dickerson dodecamer is not supported by any experimental data. In poly(dA).poly(dT), NOE and Raman studies indicate that the dA and dT units are conformationally equivalent and display the (anti-S-type sugar)-conformation; incorporation of this nucleotide geometry into a double helix leads to a conventional regular B-helix in which the width of the minor groove is 8A. The derived structure is consistent with all available experimental data on poly(dA).poly(dT) obtained under solution conditions. In the crystal structure of the dodecamer, the dA and dT units have distinctly different conformations-dA residues adopt (anti, S-type sugar pucker), while dT residues belong to (low anti, N-type sugar pucker). These different conformations of the dA and dT units along with the large propeller twist can be accommodated in a double helix in which the minor groove is shrunk from 8A to less than 4A. In the conventional right handed B-form of poly(dA).poly(dT) with the 8A wide minor groove, netropsin has to bind asymmetrically along the dA strand to account for the NOE and chemical shift data and to generate a stereochemically sound structure (Sarma et al, J. Biomole. Str. Dyns. 2, 1085, 1985).

Left-Handed Intercalated DNA Double Helix: Rendezvous of Ethidium and Actinomycin D in the Z-Helical Conformation Space

It is now very well recognized that the DNA double helix is conformationally pluralistic and that this flexibility is derived from internal motions due to backbone torsions. But what is less apparent is that such internal motions can occur in a correlated fashion and express themselves in a wide variety of structural motifs and phenomena. For example, flexibility inherent in the DNA molecule can lead to a family of Z-DNA, LZ1 and LZ2 being the two extremes and correlated internal motion can cause LZ1 in equilibrium LZ2 transition. More interestingly, such motions manifest themselves as breathing modes on the DNA lattice resulting in the sequence specific intercalation sites. Following a detailed stereochemical analyses we observed that the intercalation site for ethidium is located at the dCpdG sequence of the intercalated LZ1 helix (LZ1*) while that for actinomycin D is located at the dGpdC sequence of the intercalated LZ2 helix (LZ2*). From the stereochemistry of the drug binding we make experimentally testable predictions which are in fact supported by a few recent experimental studies. These studies also show that a left-handed intercalated B-DNA model is a viable intermediate in the Z to B transition which can hold the drug with binding energy comparable to that of the intercalated right-handed B-DNA.