Exploring DNA Structure

Week 10

Nucleic Acids

The goal for these exercises is to gain an appreciation for nucleic acid structure and its flexibility. You will begin with an analysis of the structure and dynamics of 5' mononucleotides and proceed to analysis of B and Z-DNA.

Part I

a) Get into Insight

File ® Restore folder-nucleotides

Restore object by typing guanine

Analyze the structure of the nucleotide. Measure the c (O4'-C1'-N1-C4) and d dihedral angles (C5'-C4'-C3'-O3') using the dihedral selection under the angle program on the Measure pulldown (In order to get a better idea of what the angles measure, eclipse the two central atoms of the four defining the angle.)

Rotate the sugar so that the O4', C4' and C1' are in a plane. What is the conformation of the sugar? Is this a nucleotide most likely to be found in A or B-type double helices? What is the general configuration of the glycosyl torsion angle, anti or syn?

Measure the distance from the nitrogen of the N2 amino group to each of the phosphate oxygens.

b) Open module Biopolymer

Choose Modify® Hydrogens. Select guanine as the Assembly/Molecule. Accept all defaults by clicking on Execute

Choose Potentials® select guanine as the Assembly/Molecule

Fix potentials (i.e., click on Fix button in each of the subwindows)

Execute

Move the molecule to the left third of your screen and turn it so that you may more easily see the relative orientations of the base and sugar.

c) Open module Discover_3

Choose System® Setup

Click the Expert button ON

select guanine as the Assembly/Molecule

Execute

Choose Calculate® Minimize

Set Max Steps to 10000

Turn OFF Steepest Descent AND Newton

Set Convergence to 0.1

Execute

Choose Calculate®Dynamics

Set time to 50 fs

Set Temp to 500

Execute

Choose Calculate® Minimize

Set Max Steps to 100000

Choose D_Run® Run

select guanine0 as the job

Execute

What happened to the c and d angles. Is the glycosyl torsion angle anti or syn? What is the sugar pucker?

d) Open module Biopolymer.

Modify® Geometry-of c dihedral to 100°

Record the distance between nitrogen of the N2 amino group and phosphate oxygens.

Open module Discover_3

Choose D_Run® Run

select guanine1 as the job

Execute

What happened to the glycosyl (c ) torsion angle? What happened to the sugar pucker? What do you think drove the molecule to assume this configuration? Test your hypothesis by repeating the exercise by first deleting the object guanine, restoring the object adenine from the nucleotides folder and repeating part d) after fixing the potentials on adenine. Why is this a good test of your hypothesis?

DELETE ALL OBJECTS

Part II

a) Open module Biopolymer

Under Nucleotide®Append Use this command to build a decamer (10 base pairs) of B-form duplex DNA. Notice you can click in the nucleotide value aid at the bottom of the control panel to get a list of base pairs to be used in constructing your DNA. In constructing your molecule, be sure to use at least two different types of base pairs. Before you begin you must name your molecule. (e.g., Fred or Bob).

What is the displacement of the axis of the base pair from the helical axis in this DNA? What orientation of the double helix allows you to view the displacement?

Measure and record the C1'-C1' distance across the helix for two different base bairs. Are they the same? Measure and record the c and d angles of four adjacent nucleotides on both strands near of the central base pairs. How does these values (C1'-C1' distance, c , d ) tell you about what the repeating unit of B-DNA is? (Hint-The answer to this question will become clearer when you make a similar comparison with Z-DNA helices).

Measure and record the distance between two adjacent phosphates on the same chain.

Locate the major and minor grooves. Rotate the molecule about the helical axis, while following the major or minor groove around the DNA. Measure the widths of each of these grooves. Which of these grooves might be large enough to accommodate an a -helix?

b) DELETE THIS OBJECT and build a tetramer of B-form DNA by:

Under Nucleotide® Append (please use at least two different types of base pairs).

Under Forcefield® choose Potentials and fix them by clicking on Fix in each area of the control panel. Before clicking Execute make sure the molecule pick level is set to your molecule's name and that you have this name in the Molecule Spec value aid.

Measure the P-P distance on a single strand and across the major and minor grooves, measure a base pair H-bond of one or two of the central base pairs of your tetramer. Measure the distance between complementary groups (e.g. C=O---H-N) on bases on the same strand.

c) Open module Discover_3

Choose System® Setup

Ensure the Expert button is ON (it should already be on)

In the Parameter window, set the Assem/Mol Level to Assembly

Select your <moleculename> as the Assembly/Molecule

Execute

Choose Calculate® Dynamics

Set Run time to 700 fs

Execute

Choose Analyze® Output

Click on dynamics in the Parameter window

Select File type History

Execute

Choose D_Run® Run

select <moleculename> as the job

Execute

Observe the molecular motions. What happened? How do you explain this?

d) Open molecule Analysis.

Under Trajectory®choose Get® find your <Molecule Name.his> and select it and Execute

Under Trajectory® choose Construct_Graph->New <Execute>

Plot Frames on the X-axis <Execute> and then plot Geometry dihedral on the y-axis by selecting geometry and then clicking on dihedral in the value aid box <Execute>. Define the dihedral using the atoms measured by the e angle for one of the CENTRAL BASES (see handout for definition) on the y-axis. Click on the atoms to select them.

Select End graph, then Execute and a plot will appear.

Trajectory Construct_Graph® SET NEW TO OFF! Execute

Plot Frames on the X-axis and plot Dihedral on the y-axis. This time plot the z angle for the same nucleotide as you did above. Select end graph and then Execute. A plot of the two angles over time should appear.

Is there a relationship between the phasing of the variations in these two angles? (Because the excursions of the two angles may be small, you can expand the y-axis scale by going under Graph® Scale Axis® y-axis, and setting the scale factor to 2). To determine whether the relationship you see is important or not, add a third angle to your plot by repeating the procedure you did to add z , using another angle (e.g., d , a , or b ) To understand the reason for the phasing of e and z angles, please see Fratini, et al.

Feel free to look at any other relationships between different angles, angles and energy as you wish.

DELETE ALL OBJECTS

Part III

a) Open module Biopolymer

Nucleotide® Append® A-Type DNA Duplex. Use this command to build a decamer of A-form duplex DNA. In constructing your molecule, be sure to use at least two different types of base pairs. Name your molecule. (What about Bob?)

What is the displacement of the axis of the base pair from the helical axis? What orientation of the double helix allows you to look at this?

Measure the c and d angles of one nucleotide of the central base pairs. How do these compare with the values you obtained from the B-type helix? Which of these angles serves as a measure of the main difference between the structures of the nucleotides in A and B-type helices?

Measure and record the distance between two adjacent phosphates on the same chain. How do these differ from the values obtained from the B-helix? How might this difference contribute to the smaller twist angle of A vs. B type helices.

Locate the major and minor grooves. Measure the width of the grooves. Observe how deep and narrow the major groove is. Compare this to the depth and width of the minor groove. Could an a-helix fit in either of these grooves?

DELETE THIS OBJECT

Part IV

a) Open module Biopolymer

Nucleotide® Append® Z-Type DNA Duplex. Use this command to build a decamer of Z-form duplex DNA. In constructing your molecule, be sure to use alternating GC and CG base pairs. Name your molecule.

What is the displacement of the axis of the base pair from the helical axis?

Note the striking left-handed nature of the Z-DNA double helix.

Measure the C1'-C1' distance of the four central base pairs. Measure the c and d angles of four adjacent nucleotides in the four base pairs at the center of the molecule. How do these compare with the values you obtained from the A and B-type helices? How does this distance and the values of the c and d angles tell you about what the repeating unit of Z-DNA is?

Locate the major and minor grooves. Which of these grooves might be large enough to accommodate an a -helix? Is one of them really a groove at all???

DELETE THIS OBJECT

Exit from insightII

Before logging out, please delete the files you created by the minimization and dynamics runs. For example, to do this type

rm gua*.* (=remove all files that have names starting with gua as the first three letters)

Repeat this with the adenine and your <tetranucleotide> name files.

LOGOUT AND GO HOME!