Moss Tom. DNA-protein interactions: principles and protocols

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Circular Dichroism 503

503

From:

Methods in Molecular Biology, vol. 148: DNA–Protein Interactions: Principles and Protocols, 2nd ed.

Edited by: T. Moss © Humana Press Inc., Totowa, NJ

Circular Dichroism for the Analysis

of Protein–DNA Interactions

Mark L. Carpenter, Anthony W. Oliver, and G. Geoff Kneale

1. Introduction

The asymmetric carbon atoms present in the sugars of nucleotides and in all

the amino acids (with the exception of glycine) results in nucleic acids and

proteins displaying optical activity. Further contributions to the optical activ-

ity of the polymers result from their ability to form well-defined secondary

structures, in particular helices, which themselves possess asymmetry. As a

consequence, circular dichroism (CD) has found widespread use in secondary

structure prediction of proteins (1). Similar studies, though less widespread,

have sought to correlate structural parameters of DNA with their CD spectrum

(2), with some success particularly in assigning quaternary structures to nucleic

acids (e.g., in the case of DNA triplexes and G-quartet mediated structures)

(3,4). It follows that the disruption of secondary structure by, for example,

denaturation or ligand binding can be usefully followed by circular dichroism.

Plane polarized light can be resolved into left- and right-handed circularly

polarized components. Circular dichroism measures the difference in the

absorption of these two components,

∆ε = ε

L –

(1)

where ε is the molar extinction coefficient (M

–1

/cm

–1)

for the left (L) and right

(R) components (see Note 1). When passing through an optically active sample,

the plane of polarized light is also rotated, which means that the emerging

beam is elliptically polarized. Thus, CD is often expressed in terms of elliptic-

ity (θ

, in degrees) or molar ellipticity ([θ]

, in degrees cm

/dmol).

[θ]

= 100 θ

/cl (2)

504 Carpenter, Oliver, and Kneale

where c is the molar concentration and l is the pathlength (in cm). The two

expressions are interconvertible via the expression [θ]

= 3300 ∆ε.

The overlap of the ultraviolet (UV) absorption bands of nucleic acids and

proteins means that CD studies of protein–DNA interactions can be compli-

cated by the contributions observed from both components. This is particularly

true for wavelengths less than 250 nm. In practice, CD spectra between 250

and 300 nm are dominated by that of the nucleic acid, the contribution arising

from the aromatic chromophores of the protein being weak by comparison.

Changes in conformation can usually be attributed to the polynucleotide, as the

random distribution of aromatic amino acids means a large conformational

change throughout the protein would be required to cause a significant change

in the CD spectrum.

The low-molar ellipticity of polynucleotides means that for accurate CD

measurements, high concentrations (10

–4

M to 10 M) of nucleotide are required.

For this reason, circular dichroism is not generally used to determine binding

constants of protein–DNA interactions. However, circular dichroism can be

used to obtain accurate values for the stoichiometry of protein–nucleic acid

interactions (5), and in the case of the bacteriophage, the fd gene 5 protein was

used to show the existence of two distinct binding modes (6). Circular dichro-

ism has also been used to show that conformational changes induced by the

bound Lac repressor are different for operator DNA and for random sequence

DNA (7). Similar studies on the Gal repressor demonstrated the involvement

of the central G-C base pairs of the operator sequence in repressor-induced

conformational changes (8). Studies on the interaction of the cro protein of

bacteriophage λ have also revealed different conformational changes for specific

and nonspecific DNA binding (9). Despite the apparent lack of any direct inter-

action of the central base pair of the operator sequence with the cro protein, base

substitution at this site was shown to affect the CD spectrum considerably.

Some additional examples in which CD has been used to examine protein–

DNA interactions include the SRY-related protein Sox-5 (10), the type IC DNA

methyltransferase M. EcoR124I (11), and the bacteriophage Pf3 single-

stranded DNA-binding protein (ssDBP) (12).

It should be emphasized that circular dichroism provides complementary data to

other spectroscopic techniques such as fluorescence, because each technique can

monitor different components of the interaction. For this reason, even the apparent

stoichiometry of binding estimated by each technique could be significantly

different despite it being measured under the same solution conditions (5).

2. Materials

1. A high-quality quartz cell with low strain is required for accurate measurements.

The cell pathlength will depend on the absorption properties and concentration of

Circular Dichroism 505

the sample. Cells with pathlengths between 0.05 cm and 1 cm are often used,

depending on the CD signal to be measured and the absorption of the sample

(including the buffer). A pathlength of 1 cm is usually recommended for mea-

surement of the DNA signal in the vicinity of 275 nm, where the signal is weak

and buffer absorption is negligible.

2. Buffers should be prepared using high-quality reagents and water. Use buffers

that have low absorbance in the wavelength region of interest. Tris-HCl, perchlo-

rate, and phosphate are routinely used.

3. Stock solutions of appropriate protein and nucleic acid solutions in the same

buffer. The protein should be as concentrated as possible to minimize dilution

during the titration. If a synthetic DNA fragment containing the recognition

sequence is to be used, it should be close to the minimum size required for bind-

ing to maximize the change in CD signal. To avoid denaturation and degradation,

keep concentrated solutions of protein and DNA frozen in small aliquots, assum-

ing it has been established that this procedure does not damage the protein.

4. A supply of dry nitrogen (oxygen free).

5. (+)10-Camphor–sulfonic acid at a concentration of 0.5 mg/mL, used as a calibra-

tion standard. Check the accurate concentration by UV spectroscopy using a

molar extinction coefficient of 34.5 at 285 nm (1).

6. A circular dichroism spectrometer (spectropolarimeter). We currently make use

of a Jasco J720 spectrometer.

3. Methods

For most proteins, there is only a weak signal from aromatic amino acids in

the region of the CD spectrum between 250 and 300 nm compared to that seen

for nucleic acids. Experiments involving the addition of protein can thus be

conveniently carried out in this wavelength range, as described in the follow-

ing. Below 250 nm, both proteins and DNA have optical activity and any

experiments here may require resolution of the spectrum into protein and DNA

components (see ref. 13 for an example of where a mutant protein has been

used to assign and interpret overlapping CD bands).

1. To prevent damage to the optics, flush the instrument with nitrogen for 10–15 min

before switching on the lamp. Continue to purge the instrument for the duration of

the experiment (see Note 2).

2. Switch on the lamp and allow the instrument to stabilize for 30 min before mak-

ing any measurements.

3. While waiting for the instrument to warm up, measure the UV spectrum of

both the DNA and protein and calculate the concentration of the stock solu-

tions from their extinction coefficients. The stock solution of protein should be

as high a concentration as possible, to minimize corrections for dilution in subse-

quent titrations.

4. Measure the UV absorbance of the cell to be used in the CD experiment against

an air blank (see Note 3).

506 Carpenter, Oliver, and Kneale

5. Using the data from steps 3 and 4, determine the concentrations of DNA and

protein that can be used in the experiment such that the total absorbance of all

components including the cell is <1.0.

6. Once the CD instrument has warmed up, use a 1-cm cell filled with water to

determine the baseline spectrum between 250 and 320 nm (see Note 4). This

should be flat.

7. Calibrate the instrument by replacing the water with the previously prepared

solution of camphor sulfonic acid and measure the CD between 250 and 320 nm.

A 0.5-mg/mL solution in a 1-cm pathlength cell has ellipticity of 168 mdeg at

290.5 nm.

8. Take the cell set aside for the experiment and fill with buffer. Place the cell in the

instrument, taking care to note the orientation of its faces in the beam. If using a

cylindrical cell, place it so that the neck of the cell rests against the side of the

cell holder. Run a baseline spectrum between 250 and 320 nm.

9. Replace the buffer with the DNA solution and run the spectrum under the same

conditions. If you remove the cell to do this, remember to place the cell back in

the holder with the same face toward the light source.

10. Accurately pipet a small aliquot of the stock protein solution into the DNA in the

cell and mix. Allow time for equilibration and measure the CD spectrum. The

volume of the protein solution added will be determined by its concentration,

the DNA concentration in the cuvet, and a rough idea of the expected stoichiom-

etry. For initial experiments, the molar quantity of protein added at each step

should be perhaps 10% of that of the DNA.

11. Repeat the addition of protein to the DNA until no further changes in the CD

are observed.

12. Thoroughly wash the cell and refill with buffer. Rerun the baseline spectrum. If

the baseline has drifted from its initial value, this will have to be considered

when analyzing your results. If drifting is severe, see Note 5.

13. The collected data will need to be processed (i.e., to remove the CD signal due to

buffer components and/or to correct baseline values) (see Note 6.)

14. Plot the measured CD parameter at a particular wavelength against the concen-

tration of protein added. The stoichiometry can be determined from the point at

which a line drawn along the initial slope intersects that of the titration end point,

which should be horizontal assuming dilution (if significant) has been corrected

for (see Fig. 1).

15. Once the spectral changes and stoichiometry have been established, it is often

useful to repeat the experiment with rather more titration points, using smaller

Fig. 1. (opposite page) Circular dichroism titrations of Pf1 gene 5 protein to poly

dT. In this example, the protein is a single-stranded DNA-binding protein that binds

cooperatively to DNA with little sequence preference. (A) Successive CD spectra

during the titration of poly dT with Pf1 gene 5 protein. The top curve corresponds to

free DNA; below are increasing concentrations of protein corresponding to molar

ratios of nucleotide to protein of 19.2, 6.7 and 4.0 (which represents saturation).

Circular Dichroism 507

(B) The normalized change in CD signal at 276 nm is plotted against the ratio of gene

5 protein:poly(dT) (expressed as subunit concentration:nucleotide). The data show

clear stoichiometric binding with an end point of 0.25 (i.e., 4.0 nucleotides bound per

protein subunit).

508 Carpenter, Oliver, and Kneale

aliquots of protein. This can be done at a single wavelength, without the need for

scanning. Although the maximum CD signal from DNA is normally obtained

around 275 nm, one should work at the wavelength that corresponds to the larg-

est difference between free and bound DNA, which may well be different.

The CD spectrum of the DNA at saturation can be used to assess conforma-

tional changes that result from protein binding to a given sequence. Although

analysis of the spectrum in terms of molecular structure is not straightforward

(2), it may be possible to interpret CD spectra of double-stranded DNA in terms

of changes in helical twist angle (underwinding or overwinding of the helix).

The spectral changes that accompany protein binding to different DNA

sequences or with a variety of cofactors can also be informative.

4. Notes

1. For proteins and polynucleotides, molarity is sometimes expressed in terms of

moles of amino acid or nucleotide residue respectively for such calculations.

2. High-intensity UV radiation converts oxygen to ozone, which damages the optics.

Failure to purge will lead to deterioration in instrument performance.

3. For most experiments at these wavelengths, cells with pathlengths of 0.5 cm

or 1 cm can usually be used. Longer-pathlength cells for work with more dilute

solutions are available at a price.

4. A number of instrument parameters will normally need to be set before spectra

can be recorded. For the Jasco J-700 series, these typically include slit width, CD

scale, wavelength range, step resolution, scan speed, response time and number

of accumulations. Typically, the slit width is set to 1 nm, although for high-sen-

sitivity (i.e., low CD signal) measurements, it can be increased to 2 nm (thus

improving the signal-to-noise ratio). The CD scale is set to reflect the expected

CD of the sample being measured (e.g., 0–50 mdeg). Similarly, the wavelength

range is set to cover the region required for the experiment. We typically use a

step resolution of 0.2 nm (which allows a maximum wavelength range of 400 nm

to be covered) and a scan speed of between 20 and 100 nm/min. The response

time is set in accordance with the scan speed (e.g., for a scan speed of 20 nm/min

the response time is set to 2 s [for 100 nm/min an appropriate response time

would be 0.5 s]). It is advised that the manufacturer’s recommendations be fol-

lowed when setting both scan speeds and response times, as the quality of the

data collected (in terms of the signal-to-noise ratio) can be adversely affected if

incorrect or incompatible settings are used. Between three and five accumula-

tions are generally recorded (although more can be collected), the resulting aver-

aged data offering an improvement in the CD signal-to-noise ratio.

5. If significant drifting of the baseline has occurred, the experiment may have to be

repeated. To minimize the effect of drifting, use two cells, one as a baseline ref-

erence and one containing sample. The baseline can then be standardized at each

point in the titration. Remember that when swapping cells, it is vitally important

to present the same section of the cell to the beam each time.

Circular Dichroism 509

6. The CD software supplied by Jasco allows simple calculations/derivations to

be performed with collected data (e.g., the subtraction of CD signal as a result

of the sample buffer, subtraction of a constant factor, or conversion between

standard units (millidegrees to molar ellipticity). If the signal-to-noise ratio

is particularly problematic, data can also be treated with a number of

smoothing or noise reduction functions; although if the data collection

parameters are correctly set up and the sample is at a sufficient concentration,

these procedures should not be necessary. A further useful function of the

Jasco CD software allows the collected data to be “dumped” and imported

into other software packages (e.g., Microsoft Excel or other spreadsheet/

graphing packages, which may be more familiar to the user).

References

1. Johnson, W. C., Jr. (1990) Protein secondary structure and circular dichroism: a

practical guide. PROTEINS: Struct. Funct. Genet. 7, 205–214.

2. Johnson, B. B., Dakl, K. S., Tinoco, I., Jr., Ivanov, V. I., and Zhurkin, V. B.

(1981) Correlations between deoxyribonucleic acid structural parameters and cal-

culated circular dichroism spectra. Biochemistry 20, 73–78.

3. Gray, D. M., Hung, S. H., and Johnson, K. H. (1995) Absorption and circular

dichroism spectroscopy of nucleic acid duplexes and triplexes. Methods Enzymol.

246, 19–34.

4. Hardin, C. C., Henderson, E., Watson, T., and Prosser, J. K. (1991) Monovalent

cation induced structural transitions in telomeric DNAs: G-DNA folding interme-

diates. Biochemistry 30, 4460–4472.

5. Carpenter, M. L. and Kneale, G. G. (1991) Circular dichroism and fluorescence

analysis of the interaction of Pf1 gene 5 protein with poly(dT). J. Mol. Biol. 27,

681–689 .

6. Kansy, J. W., Cluck, B. A., and Gray, D. M. (1986) The binding of fd gene 5

protein to polydeoxyribonucleotides: evidence from CD measurements for two

binding modes. J. Biomol. Struct. Dynam. 3, 1079–1110.

7. Culard, F. and Maurizot, J. C. (1981) Lac repressor–lac operator interaction.

Circular dichroism study. Nucleic Acids Res. 9, 5175–5184.

8. Wartell, R. M. and Adhya, S. (1988) DNA conformational change in Gal repres-

sor–operator complex: involvement of central G-C base pair(s) of dyad symme-

try. Nucleic Acids Res. 16, 11,531–11,541.

9. Torigoe, C., Kidokoro, S., Takimoto, M., Kyoyoku, Y., and Wada, A. (1991)

Spectroscopic studies on lambda cro protein–DNA interactions. J. Mol. Biol. 219,

733–746.

10. Connor, F., Cary, D., Read, C., Preston, N. S., Driscoll, P. C., Denny, P., et al.

(1994) DNA binding and bending properties of the post-meiotically expressed

Sry-related protein Sox–5. Nucleic Acids Res. 22, 3339–3346.

11. Taylor, I. A., Davis, K. G., Watts, D., and Kneale, G. G. (1994) DNA binding

induces a major structural transition in a type I methyltransferase. EMBO J. 13,

5772–5778.

510 Carpenter, Oliver, and Kneale

12. Powell, M. D. and Gray, D. M. (1995) Role of Tyr-22 in the binding of Pf3 ssDNA

binding proteins to nucleic acids. Biochemistry 34, 5635–5643.

13. Mark, B. L. and Gray, D. M. (1997) Tyrosine mutant helps define overlapping CD

bands from fd gene 5 protein–nucleic acid complexes. Biopolymers 42, 337–348.

General Reading

1. Bayley, P. M. (1973) The analysis of circular dichroism of biomolecules. Prog.

Biophys. 27, 1–76.

2. Bush, C. A. (1974) Ultraviolet spectroscopy, circular dichroism and optical rota-

tory dispersion, in Basic Principles in Nucleic Acid Chemistry, vol. 2 (Ts’O,

P.O.P., ed.), Academic, New York, pp. 91–169.

3. Drake, A. F. (1994) Circular dichroism. Methods Mol. Biol. 22, 219–44.

4. Gratzer, W. B. (1971) Optical rotatory dispersion and circular dichroism of nucleic

acids, in Procedures in Nucleic Acid Research, vol. 2 (Cantoni G. L. and Davies

D. R., eds.), Harper and Row, New York, pp. 3–30.

5. Woody, R. W. (1995) Circular dichroism. Methods Enzymol. 246, 34–71.

Calorimetry of Protein–DNA Complexes 511

511

From:

Methods in Molecular Biology, vol. 148: DNA–Protein Interactions: Principles and Protocols, 2nd ed.

Calorimetry of Protein-DNA Complexes

and Their Components

Christopher M. Read and Ilian Jelesarov

1. Introduction

The many detailed, though static, structures of protein–nucleic acid com-

plexes have revealed the underlying structural determinants of the binding pro-

cess: a high surface complementarity and a precise orientation of interacting

groups. However, another important question not yet fully answered is why the

molecules interact with each other. To answer such a question means that we

have to rationalize a structure energetically. This requires a deconvolution of

the relatively large negative value of the free energy of association (∆G) into

its enthalpic (∆H) and entropic (∆S) contributions. Only then can one appreci-

ate the true nature of the forces that drive the interaction of protein and nucleic

acid. Despite some promising theoretical developments and the steady accu-

mulation of experimental data, the deconvolution of ∆G into its components

remains a difficult task

Proteins and nucleic acids behave cooperatively and often undergo struc-

tural rearrangements on associating. These changes range from subtle adjust-

ments of dihedral angles to rearrangements in the orientation of domains and

even the refolding of entire binding domains. Many DNA-binding domains are

either very flexible or partly unstructured or even fully unfolded in isolation

and become folded only when bound to the specific DNA target site (1). The

DNA itself sometimes undergoes large structural deformation upon complex

formation (2–5). In energetic terms, the view that binding specificity is simply

the accumulation of specific favorable interactions between rigid binding part-

ners no longer holds and conformational changes in the two components as

well as solvent rearrangement in the binding site play an important role. Thus,

512 Read and Jelesarov

there is a complicated energetic profile involving changes in ∆G, ∆H, and ∆S

on going from the free components to the final complex.

This chapter focuses on the practical aspects of measuring the energetics of

binding of DNA-binding domains to short DNA duplexes. As an example, we

will consider the HMG domain of mouse Sox-5, hereafter referred to as just

Sox-5, as it illustrates the importance of structural changes that accompany

complex formation (6). HMG domains contain three α-helices arranged into

an L-shaped fold. Nuclear magnetic resonance (NMR) derived structures of

the HMG domain of human SRY bound to an 8-bp DNA duplex (4) as well as

the HMG domain of mouse LEF-1 complexed to 15 bp DNA (5) show that the

domain binds almost exclusively into a widened minor DNA groove. A large

bend with considerable base unstacking is introduced into the DNA, in part as

a result of the partial intercalation of an amino acid side chain (Ile in SRY and

Met in LEF-1).

The interacting components, the free Sox-5 protein and the DNA duplexes

have been examined and both structures are seen to depend significantly on

temperature (7,8). A practical problem then arises because the temperature

dependence of the enthalpy of binding, as measured by isothermal titration

calorimetry (ITC), can no longer be interpreted as being solely the result of a

unique heat capacity increment, ∆C

. As a result, the energetics of complex

formation require a thorough understanding of the energetics of the free com-

ponents (i.e., the study of the heat capacity C

, of the complex, and its free

components over a broad temperature range) as can be directly obtained by

differential scanning calorimetry (DSC). We thus wish to emphasize that for

the study of the energetics of protein–nucleic acid complexes the combined

use of ITC and DSC is necessary, because the DSC data are essential to correct

the data obtained by ITC.

1.1. The Energetics of Biomolecular Interaction

To characterize the thermodynamics of a binding reaction, it is necessary to

determine the association free energy ∆G and its enthalpic and entropic com-

ponents, ∆H and ∆S, at a given reference temperature. The heat capacity incre-

ment on binding, ∆C

, is required to predict the change of the above three

quantities with temperature, according to the general thermodynamic relation-

ships. Values for ∆G, ∆H, and ∆S may then be obtained at temperatures that

are experimentally inaccessible. Furthermore, ∆C

is a fundamental thermody-

namic quantity and contains important structural information. For example, it

has been shown that ∆C

changes with the proportion and chemical properties

of the molecular surface buried at the binding site (9,10).

∆G is accessible through different types of binding experiments because it

is related to the association constant, K

, by ∆G = –RTlnK

, where R is the gas