Application of 1D and 2D 1H NMR Spectroscopy to the Conformational Analysis of Small molecules, Oligonucleotides and Proteins.

Index:

Objectives:

To illustrate the use of nmr data in structural analysis
  1. Analysis of the conformation of a bicyclic ring system via interpretation of the 3J coupling constants using the MacroModel program (< 3 hours)
  2. Analysis of the the three-dimensional structure and base sequence of the oligonucleotide CGCGTTTTCGCG (< 3 hours)
Back to index
Back to index

Conformational Analysis of a Bicyclic Ring Compound.

The synthesis of the following compound was recently reported to be remarkably stereospecific (E.Wenkert and S. R. Piettre, J. Org. Chem., 1988, 53, 5850; compound 22 in this reference). The stereochemistry of the methoxy group was deduced from observation of a nuclear Overhauser effect between this group and the ester methyl. The 3J couplings from both H5 to H4 and H7 to H8 (2-4Hz) were reported but not analysed. It is possible to derive this and further information by application of the Karplus relationship to molecular models of this compound?

Analyse the three dimensional structure of this compound using the MacroModel program, using the coupling constant feature described in the procudure below. What conclusions regarding the stereochemistry and conformation of this system can be made? Discuss the factors that may control the conformational stability of this compound, and comment on whether the original authors may have missed anything!

Procedure

Use the MacroModel program and from the READ menu, enter the following file names, exactly as shown below, lower case and all; 
/usr/local/chorg/ugteach/chair
/usr/local/chorg/ugteach/boat-high
/usr/local/chorg/ugteach/boat-low
Enter the ANLYZ menu in MacroModel, then select NMR, and finally COUPL. With the left mouse button, click at two hydrogen atoms whose nmr coupling constant you want to predict. The MacroModel program calculates the dihedral angles between these hydrogens, applies the appropriate Karplus relationship (for which a reference is given) and displays the expected value. As it happens, one conformation fits the observed nmr data almost exactly. There may be other, maybe even better conformations. If you want to re-minimise new conformations, follow the instructions given below.
Back to index

Sequencing an Oligonucleotide. Two-dimensional NOESY Spectroscopy

The bases guanine (G), cytosine (C), adenine (A) and thymine (T) contain very simple aromatic proton spin systems. When an oligomer is built up from the corresponding nucleotides, these proton spin systems do not interact via any form of proton-proton coupling. Exactly the same is true of the basic proton nmr of amino acids and their oligomers, peptides and proteins. However, conventional 1D nmr spectra recorded for such molecules tend to be very complex, largely because of the considerable overlap between the spectra of each component nucleotide or amino acid, and the superposition of chemical shift (d) and coupling constant (J) information on the same axis. One way of reducing the complexity is to separate the d and J axes into a 2D plot (knowns as a COSY spectrum), from which much useful information about the assignment of the individual bases or amino acids can be deduced. More problematic is determining the precise structural relationships between individual nucleotides or amino acids in solution. Conventional 1D nmr methods provide no data describing the relationship between different amino acids or nucleotides since there no simple coupling constant information to use. One nmr phenomenon known as the nuclear Overhauser effect (nOe) provides exactly the missing information about the proximity in space of protons rather than the through-bond connectivity information provided by conventional coupling constants.

The nOe effect is manifested as a change in the intensity (typically between 1% and 20%) of one or more resonances in the nmr spectrum as a result of rf irradiation at a second resonance. The magnitude of the effect depends upon the distance in space between the two nuclei and is proportional to 1/r6, where r is the internuclear separation. In practical terms, nOes are rarely seen between pairs of protons that are separated by more than about 4.5Å, but in a large protein or fragment of DNA, there may be several hundred such 'non-bonded' contacts which taken together may provide sufficient information to infer the three-dimensional structure. If the nOe experiment is carried out in a 1D sense, the change in intensity is superimposed upon the spectrum itself, and spectral subtraction techniques (NOEDS; nOe difference spectra) have to be employed to render the nOe visible. An alternative way of separating the nOe effect from the spectrum is to use a 2D technique in which the spectrum appears along a diagonal and the nOe is revealed as cross-peaks off the diagonal. Shown below is a hypothetical NOESY spectrum for a molecule containing four protons A-D. Protons C and B are close, C and D are further apart and A is distant from all the other protons.

All four protons give rise to a peak on the diagonal of the 2D spectrum (A-D). The position of this peak corresponds to the chemical shift of the peak which would be observed in a normal 1D nmr spectrum. The nOes are seen as 'cross-peaks'which are symmetrical about the diagonal. The intensity of the cross peak is approximately proportional to the distance between the two protons, although several other factors not related to distance can also control the intensity. From this, we can tell that B and C are probably closer together than C and D are. No information about A can be obtained from this spectrum. For a large macromolecule, frequently several hundred cross peaks are visible, and provided the diagonal elements can be assigned to specific bases or amino acids, the three-dimensional structure can be determined.

To illustrate the latter aspect, a 2D NOESY spectrum for the oligonucleotide CGCGTTTTCGCG (a single DNA strand containing a TTTT loop and four terminal complementary pairs) will be analysed to show how the sequence of base pairs can be determined from nmr spectroscopy. The spectrum below (from  D. R. Hare, B. R. Reid, Biochemistry, 1986, 25, 5341; A. Pardi, D. R. Hare, C. Wang, Proc. Natl. Acad. Sci., 1988, 85, 8785) shows a cross-peak connectivity map with nOe effects between the chemical shift region 8.0 - 7.2 (the aromatic protons labelled HC, HG or HT) and 6.1 - 5.3 ppm (the ribose component HR).

To view a 3D model of the DNA, click on the black square showing the molecule in the spectrum below. If you move the mouse over each peak, its assignment will appear in the information box on the bottom of the browser.

The proximity of the aromatic protons of the bases to the anomeric protons of the ribose rings is a typical feature of the helical structure of DNA fragments. Normally, individual protons display more than one nOe effect, and such a 'stepping stone' approach can serve to establish the base sequence. In this example, two exactly vertical nOe peaks would indicate that an individual anomeric proton was close to two different aromatic base protons, one from its own base, and one from an adjacent base in the oligomer. From such information a partial or complete base sequence can be mapped.

You should follow the horizontal/vertical connectivity displayed in the 2D NOESY spectrum above and analyse the features in your laboratory report. You should for example discuss if a continuous connectivity can indeed be established for all 12 nucleotides, and how many 'stepping stones' are used to establish the connectivity within any one pair of nucleotides.
 

  • PROCEDURE
  • Login to a turquoise Silicon Graphics workstation  in the HHMI compute room and run the GRASP program. The DNA PDB file is here.  Using GRASP, you should inspect for possible close contacts between eg pairs of aromatic/anomeric protons on the same or on adjacent base/ribose units and relate these to the 2D NOESY spectra shown above.
    Back to index

    • Back to index
    AFTER H. S. Rzepa and ICSTM Chemistry Department, 1994-1998.