Moss Tom. DNA-protein interactions: principles and protocols

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Nitration of Tyrosine Residues 295

3. Methods

The method is a fairly general one for protein-nucleic acid complexes. How-

ever, precise details of conditions for nucleoprotein complex dissociation and

peptide mapping will vary with the system under investigation. As an example

of the technique, nitration of the Pf1 gene 5 protein and nucleoprotein complex

will be described (5).

3.1. Nitration

1. Desalt the protein or nucleoprotein complex into nitration buffer to a concentra-

tion between 0.5 and 5 mg/mL (see Note 1). For initial determination of nitrated

residues, 0.5 mg of protein should be sufficient. However, if a time-course

experiment is performed, larger amounts of protein are required.

2. To 1 mL of sample, add a 10-fold molar excess of 300 mM TNM (in ethanol) and

incubate at room temperature for 1 h, stirring gently (see Note 2). The reaction

is stopped by the addition of acid (add HCl to pH 2.0) or by rapid desalting into

10 mM Tris–HCl pH 8.0 (see Note 3).

3. Run an aliquot of the modified protein–nucleoprotein complex on an SDS gel,

together with native protein, to determine whether there has been any TNM-

induced crosslinking (see Note 4). If analyzing the free protein proceed to step 5;

if modifying the nucleoprotein complex, proceed to step 4.

4. Dissociate the nucleoprotein complex by the addition of salt (see Note 5). Large

DNA fragments can be removed by ultracentrifugation, whereas smaller frag-

ments can be either digested with nucleases or removed by gel filtration. The

protein is then dialyzed or desalted into the appropriate protease digestion buffer.

5. Digest the protein to completion with the desired protease(s) and then lyophilize

the peptides for separation by HPLC. The peptides may be stored at –20°C.

In the example provided, the Pf1 gene 5 protein and nucleoprotein complex

were incubated at room temperature in the presence of a 64 M excess of TNM

(300 mM in ethanol) for 3 h. The reaction was stopped by desalting the protein

(and nucleoprotein complex) into 10 mM Tris-HCl, pH 8.0. The nucleoprotein

complex was dissociated by the addition of MgCl

to 1 M and the phage

genomic DNA was then removed by ultracentrifugation at 221,000g (in a

Beckman L8 ultracentrifuge; 70.1 Ti rotor) for 2.5 h. The protein was desalted

into 10 mM Tris-HCl, pH 8.0 for proteolysis and digestion with trypsin (Sigma

[St. Louis, MO], TCPK treated) at an enzyme substrate ratio of 1:25 (w/w) for

3 h at 37°C. Phenylmethane sulfonyl fluoride was added to a final concentra-

tion of 1 mM and the sample was lyophilized overnight. This procedure results

in the complete separation of the three tyrosine-containing tryptic peptides.

3.2. Peptide Mapping

1. Peptides can usually be separated by reverse-phase HPLC on a C

column. Gen-

erally the peptides are applied to the column in 8 M urea and 2% β-mercapto-

296 Plyte

ethanol and separated in an acetonitrile gradient in the presence of 0.05–0.1%

TFA. The acetonitrile gradient must be determined empirically for each particu-

lar protein.

2. Determine separation conditions for peptides from the native protein (see Note

6) and identify tyrosine-containing peptides (see Note 7).

3. Apply peptides from the nitrated protein and initially elute under the same condi-

tions that were used for the native protein (see Note 8). If necessary, change the

acetonitrile gradient to achieve separation of the tyrosine-containing peptides and

their nitrated counterparts.

In the example provided, the tryptic peptides from a native gene 5 protein

were resuspended in 200 µL of 8 M urea and 2% β-mercaptoethanol and

clarified prior to HPLC analysis (Fig. 1, top). Tyrosine-containing pep-

tides were initially detected by their fluorescence properties (see Note 7) and

then identified by automated Edman degradation on an Applied Biosystems

477A pulsed liquid amino acid sequencer. Nitrated peptides were applied to

the C

column and separated under identical conditions (Fig. 1, bottom). The

nitrated peptides were initially detected by their altered retention times and by

virtue of their yellow color in 10 mM Tris-HCl, pH 8.0. The identity of the

nitrated peptides was subsequently confirmed by N-terminal sequencing (see

Note 9).

3.3. Functional Studies

As discussed in Subheading 1., one should check whether nitration of the

protein impairs the ability to bind DNA (other chapters in this volume can be

consulted for possible approaches such as EMSA [Chapter 2] or DNaseI

footprinting [Chapter 3]). The protein isolated from the nitrated nucleoprotein

complex should be checked for DNA binding. Because the target amino acid

residues in contact with the DNA should have been protected from modifica-

tion, the protein from the nitrated complex would be expected to retain DNA-

binding activity.

4. Notes

1. As an alternative to desalting, the protein can be dialyzed into nitration buffer.

2. The molar excess of TNM can be increased to ensure maximal modification (e.g.,

the Pf1 gene 5 protein was nitrated in a 64-fold molar excess of TNM in the

example provided). Note, however, that at high concentrations, protein insolubil-

ity can become a problem.

3. One can desalt the protein into a buffer appropriate for proteolysis or dissociation

of the nucleoprotein complex at this stage, as required.

4. Tetranitromethane-induced crosslinking has been widely reported, and an SDS

gel should be run to check for the appearance of adducts. Reducing the concen-

tration of portion or molar excess of TNM may help to limit adduct formation.

Nitration of Tyrosine Residues 297

Fig. 1. High-performance liquid chromatographic elution profile of tryptic peptides

of the Pf1 gene 5 protein for (top) native protein and (bottom) nitrated protein. Peaks Y21,

Y30, and Y55 correspond to tryptic peptides containing tyrosine 21, 30, and 55,

respectively. n denotes a nitrated peptide. Aliquots (100 µL) were applied to a µBondapak

C18 HPLC column (Waters Associates) (300 × 4.6 mm inner diameter) fitted with a

C18 guard column. The HPLC buffers for this experiment were as follows: buffer A:

0.05% TFA/H

O; buffer B: 0.05% TFA/acetonitrile. Peptides were separated in the

following gradient at a flow rate of 2 mL/min: O% B for 5 min; 0–10% B in 20 min;

10–55% B in 45 min; 55–90% B in 5 min; 90% B for 5 min; 90–100% B in 5 min.

298 Plyte

Gel filtration is another way of removing the adducts prior to peptide mapping

(see also Note 10).

5. Usually, protein–nucleic acid interactions can be disrupted by the addition of

NaCl or MgCl

to 1–2 M. The conditions required to effect separation will vary

with the nature of the complex.

6. It is not essential to have complete separation of all fragments, only separation of

the tyrosine-containing peptides and their nitrated counterparts. The HPLC

conditions should be adjusted to achieve this.

7. Tyrosine residues have a characteristic fluorescence emission maximum at 303 nm

when excited at 278 nm. This phenomenon can be used initially to determine

which peptides contain a tyrosine residue (this may not be possible, however, if

there is a tryptophan residue present in the same peptide as a result of energy

transfer). If on-line fluorescence detection is not available, the fractions can

be taken directly from the HPLC and analyzed in a fluorimeter. The peptides

should then be unambiguously identified by either N-terminal sequencing or

amino acid analysis.

8. The addition of a nitrate group to the tyrosine should alter the hydrophobicity

and, hence, the retention time of that particular peptide in an acetonitrile gradient.

This should allow immediate identification of the nitrated peptides. However, it

is possible that a nitrated peptide comigrates with another unmodified peptide.

Therefore, freeze-dry all peptides from HPLC and resuspend in 10 mM Tris-HCl,

pH 8.0: The nitrated peptides will have a faint yellow color (absorbance maxi-

mum at 428 nm).

9. For peptides sequenced on an applied biosystems 477A pulse liquid amino acid

sequencer (fitted with a 120A separation system for the analysis of PTH-derivitized

amino acids), PTC-3-nitrotyrosine elutes just after DTPU.

10. TNM induced oligomerization has been observed on the nitration of numerous pro-

teins including several DNA-binding proteins (4,12,13). This is usually considered

undesirable, and steps are often taken to reduce the crosslinking and remove adducts

before analysis (e.g., by gel filtration). However, advantage can be taken of this

crosslinking ability; Martinson and McCarthy (14) used TNM as a reagent to

crosslink histones specifically. On nitration with TNM, we have also shown that

the Pf1 gene 5 protein forms an SDS-stable dimer (13). Initial analysis of the pep-

tide adduct in this case suggested that tyrosine 55 from one monomer was

crosslinked to phenylalanine 76 from the other monomer (forming an interdimer

crosslink rather than an intradimer crosslink). The adducts are thought to form via

a free-radical mechanism, resulting in zero-length crosslinks between residues in

close proximity (14,15) Thus, if adduct formation is limited to one or two species,

additional structural information can be obtained from the experiment. The

crosslinked proteins should be digested and the peptide adduct purified by

HPLC. N-Terminal sequencing, amino acid analysis, and gas chromatograpy–mass

spectroscopy (of the hydrolyzed peptide) should enable unambiguous identifica-

tion of the two residues participating in the crosslink and provide structural infor-

mation concerning the relative positions of these residues in the protein.

Nitration of Tyrosine Residues 299

References

1. Mueller, M. J., Samuelson, B., and Haeggstrom, J. Z. (1995) Chemical modifica-

tion of leukotriene A4 hydrolase. Indications for essential tryosyl and arginyl resi-

dues at the active site. Biochemistry 34, 3536–3543.

2. Hawes, J. W., Crabb, D. H., Chan, R. M., Rougraff, P. M., and Harris, A. (1994)

Chemical modification and site-directed mutagenesis studies of rat 3-hydroxy-

isobuyrate dehydrogenase. Biochemistry 34, 4231–4237.

3. Kochhar, S., Hunziker, P. E., Leong-Morgenthaler, P., and Hottinger, H. (1992)

Primary strucure, physiochemical properties and hemical modification of NAD+-

dependent d-lactate dehydrogenase. J. Biol. Chem. 267, 8499–8513.

4. Anderson, R., Nakashima, Y., and Coleman, J. (1975) Chemical modification of

functional residues of the Fd gene 5 DNA-binding protein. Biochemistry 14,

907–917.

5. Plyte, S. E. and Kneale, G. G. (1991) Mapping the DNA binding site of the Pf1

gene 5 protein. Protein Eng. 4(5), 553–560.

6. Lundblad, R. and Noyes, M. (1984) Chemical Reagents for Protein Modification

I and II, CRC, Boca Raton, FL.

7. Riordan, J., Sokolovsky, M., and Vallee, B. (1967) Environmentally sensitive

tyrosine residues. Nitration with tetranitromethane. Biochemistry 6, 358.

8. Sokolovsky, M., Riordan, J., and Vallee, B. (1966) Tetranitromethane. A reagent

for the nitration of tyrosyl residues in proteins. Biochemistry 5, 3582–3589.

9. Sokolovsky, M., Harell, G., and Riordan, J. (1969) Reaction of tetranitromethane

with sulphydryl groips in proteins. Biochemistry 8, 4740–4745.

10. Dimicoli, J. and Helene, C. (1974) Interaction of aromatic redisues of proteins

with nucelic acids I and II. Biochemistry 13, 714–730.

11. Sokolovsky, M. Riordan, J., and Vallee, B. (1967) Conversion of 3-nitrotyrosine

to 3-amino-tyrosine in peptides and proteins. Biochem. Biophys. Res. Commun.

27, 20.

12. Anderson, R. and Coleman, J. (1975) Physiochemical properties of DNA-binding

proteins: gene 32 protein of T4 and Escherichia coli unwinding protein.

Biochemistry 1, 5485–5491.

13. Plyte, S. E. (1990) The biochemical and biophysical characterization of the Pf1

gene 5 protein and its complex with nucelic acids, Ph.D. thesis, Portsmouth Uni-

versity, Portsmouth, UK.

14. Martinson, H. and McCarthy, B (1975) Histone–histone associations within chro-

matin. Crosslinking studies using tetranitromethane. Biochemistry 14, 1073–1078.

15. Williams, J. and Lowe, J. (1971) The crosslinking of tyrosine with tetranitro-

methane. Biochem. J. 121, 203–209.

Modification of Lysine by Reductive Methylation 301

301

From:

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

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

Chemical Modification of Lysine

by Reductive Methylation

A Probe for Residues Involved in DNA Binding

Ian A. Taylor and Michelle Webb

1. Introduction

The basic side chains of lysine residues often play essential roles in DNA–

protein recognition. They are able to contribute to the overall affinity of an

interaction through nonspecific charge–charge interactions with the phosphate

backbone and contribute substantially to the specificity of the interaction by

forming direct hydrogen bonds with functional groups on the edges of the

bases. This dual role and their almost ubiquitous presence in the interface of

DNA–protein complexes make them very attractive targets for chemical modi-

fication experiments.

Numerous chemical reagents to chemically modify lysine side chains in pro-

teins are available (1). Unfortunately, the conditions required for such

procedures are often harsh and result in total denaturation of the protein. Fur-

thermore, many reagents are not entirely specific to lysine and often react with

other residues such as cysteine, histidine, and tyrosine. Two methods, that are

able to specifically modify lysine residues under native conditions and have

been applied successfully to the investigation of protein nucleic acid interac-

tions, are amidination with imidoesters (2) and, the subject of this chapter,

reductive alkylation (3).

Reductive alkylation has become a widespread and well-established tech-

nique for the specific modification of lysine residues and a variety of reagents

have been used to produce this chemical modification (4). A reductive

methylation reaction using formaldehyde and the reducing agent sodium

cyanoborohydride (5) is particularly useful because under mild solution condi-

302 Taylor and Webb

tions (aqueous buffer, pH 7–8), the accessible lysine residues on proteins are

completely converted to the ε-N,N-dimethyl derivatives. The reaction (Fig. 1)

occurs in two distinct phases. Initially, the ε-amino group of the lysine forms

an adduct with the formaldehyde to produce a Schiff base. This then undergoes

reduction by sodium cyanoborohydride to the monomethylamine derivative in

the second part of the reaction. A further round of the reaction, which occurs

more rapidly than the first produces the dimethyl derivative.

The attractiveness of this modification is that dimethylation of the lysine

side chain is a relatively small chemical change. The pK

is only slightly

affected and may even remain unchanged (5). Because of this, the residue main-

tains its ionization properties and the potential for the formation of the same

ion-pair interactions as in the unmodified protein remains, although there is

some loss of hydrogen-bonding capacity. Nevertheless, the modification is a

mild one and unlikely to significantly perturb the native enzyme structure.

A major use of any chemical modification procedure is the incorporation of

isotopic labels at specific positions in proteins. Reductive methylation experi-

ments that incorporate

C have been used to probe the environment of lysine

side chains in proteins using nuclear magnetic resonance (NMR) spectroscopy

(7). The incorporation of radiolabels into proteins by reductive methylation

enables the number of lysines that are accessible to be determined. In the case

of DNA-binding proteins, the labeling is carried out on the free protein and the

DNA–protein complex. This immediately provides information about the num-

ber of lysines present in the DNA-binding site. Subsequent peptide mapping

strategies allow identification of specific residues, the degree of labeling at

particular sites is then used to derive the location of that residue within the

protein. In this way, [

H] formaldehyde has been used as a source of radiolabel

to probe the role of the lysine residues of the core histones in the nucleosome

Fig. 1. Reductive methylation of lysine. The reaction scheme (6) occurs in two

distinct phases. Adduction of formaldehyde to the lysine ε-amino group generates the

Schiff base (A), which is then reduced by sodium cyanoborohydride to ε-N-mono-

methyl-lysine (B). A second round of the reaction generates the final product ε-N,N-

dimethyl-lysine (C).

Modification of Lysine by Reductive Methylation 303

(8), the interaction of the linker histone H5 with nucleosomes in long chroma-

tin (9), and the role of lysine residues in a specific DNA–protein complex

between the type I DNA methylase M.EcoR124I (10) and a short DNA duplex

containing its recognition site.

Subheading 3. contains a set of protocols to enable quantification of the

number of lysine residues susceptible to reductive methylation in a protein and

a DNA–protein complex. The peptide mapping and N-terminal protein-

sequencing procedures required to identify and determine the extent of modifi-

cation at individual residues are also described. Because the modification

reaction is sensitive to contaminants in commercial grades of sodium cyano-

borohydride (most likely cyanide), a method to recrystallize the reagent (6) is

described in Subheading 3.1. In order to obtain quantitative information about

the total extent of protein modification and/or determine the relative accessi-

bility of individual residues, it is vital that the specific activity of the [

formaldehyde be determined accurately. Subheading 3.2. describes a protocol

to do this using a simple peptide substrate α-melanocyte-stimulating hormone

(α-MSH). The data from this experiment then allow quantitative conclusions

to be drawn from the subsequent protein protection and peptide mapping

experiments that are described in Subheadings 3.3. and 3.4.

2. Materials

2.1. Reagents and Materials

1. Reagents were obtained as follows: urea, formic acid, hydrochloric acid and

glacial acetic acid (ARISTAR grade), dichloromethane (dried, AnalaR), gly-

cine, Tris (hydroxymethyl) methylamine, sodium chloride and Na

EDTA

(AnalaR), trifluoroacetic acid and water (HiPerSolv grade), acetonitrile (far

UV HiPerSolv grade), and sodium cyanoborohydride (Schuchardt), all from

Merck; dithiothreitol (DTT) and HEPES (both molecular biology grade), α-MSH

and silica gel (Type III, indicating) from Sigma [

H] formaldehyde (approx

100 Ci/mol). NEN radiochemicals; filter paper (No. 1) from Whatman; dialysis

membrane (Slide-A-Lyser) from Pierce; scintillation fluid (Ecoscint™ H) from

National Diagnostics); trypsin (sequencing grade) and Pefabloc™ from

Boehringer Mannheim.

2. DNA-binding protein (approx 10 mg).

3. Stock solution of oligonucleotide duplex, 300 µM (1 µmol synthesis, high-per-

formance liquid chromatography [HPLC] purified).

4. Formaldehyde (Sigma): 37% solution and 15% methanol stabilizer.

5. 8 M urea and 50 mM acetic acid.

6. 10 mM HEPES, pH 7.5.

7. 1 M Na-glycine, pH 7.0.

8. 50 mM TBS: 10 mM Tris-HCl, 50 mM NaCl, and 1 mM EDTA, pH 7.5.

9. 2 M Tris base.

304 Taylor and Webb

2.2. Equipment

1. HPLC: binary gradient formation using high-pressure-mixing, variable-wave-

length ultraviolet (UV) absorbance detector and fraction collection are required.

2. Reverse-phase HPLC columns: semipreparative C

(9.4 × 250 mm), analytical

(4.6 × 250 mm), and analytical C

(4.6 × 250 mm). Zorbax 300SB or an

equivalent wide-pore packing is recommended.

3. High-resolution anion-exchange column (e.g., TSK-GEL, DEAE-NPR [4.6 ×

35 mm]).

4. Freeze-dryer.

5. Scanning (UV/visible) spectrophotometer.

6. Liquid scintillation counter.

7. Protein-sequencing facilities.

3. Methods

3.1. Recrystallization of Sodium Cyanoborohydride

1. Dissolve 44 g of sodium cyanoborohydride in 100 mL of acetonitrile and remove

any undissolved material by centrifugation.

2. Add 600 mL of dichloromethane to the mixture place in a sealed container and

allow the sodium cyanoborohydride to precipitate overnight at 4°C.

3. Filter the mixture (Whatman filter paper No. 1) to collect the precipitate and

wash with an additional 100 mL of cold dichloromethane.

4. Allow the powder to dry and store in a vacuum desiccator containing silica gel.

Prepare aqueous stock solutions immediately before use.

3.2. Determination of the Effective Specific Activity

H Formaldehyde

There is a substantial variation in the effective specific activity of different

batches of [

H] formaldehyde. In order to accommodate this, it is necessary to

determine the effective specific activity of each individual batch. A convenient

way to do this is to use a simple peptide substrate for which the number of

accessible amino groups is known. The effective specific activity can then be

calculated from the amount of radioactivity that can be incorporated into

the fully modified peptide. For this purpose the peptide hormone α-MSH

(Ac-SYSMEHFRWGKPV-NH

) is recommended (see Note 1).

1. Redissolve the contents of a 1-mg vial of α-MSH in 1 mL of 8 M urea and 50 mM

acetic acid.

2. Equilibrate a C

reverse-phase column in 2% acetonitrile and 0.05% (v/v)

trifluoroacetic acid (TFA) at a flow rate of 1 mL/min and set the UV absorbance

detector to 280 nm. Apply the peptide in approx 200-µg aliquots and elute with a

0–60% increasing gradient of acetonitrile, 0.05% TFA (v/v) over 60 min.

3. Collect the peptide-containing fractions from multiple runs, pool, and lyophilize

them. Store the purified peptide at –20°C in a box containing silica gel.

Modification of Lysine by Reductive Methylation 305

4. Dissolve 100 nmol of purified α-MSH in 0.5 mL of 10 mM HEPES, pH 7.5. Add

10 mM sodium cyanoborohydride (freshly made) followed by 2 mM [

formaldehyde (see Notes 2 and 3) and incubate at 24°C for 3 h. After this time

add a second aliquot of the reagents and allow the reaction to continue for an

additional 3 h.

5. Adjust the sample to 8 M urea and 50 mM acetic acid and purify the modified

peptide by reverse-phase chromatography using the same gradient as in step 2.

Pool the fractions, lyophilize, and store at –20°C in a box containing silica gel.

6. To determine the specific activity of the modified peptide, redissolve the labeled

peptide in 1 mL of water and determine its concentration from the absorbance at

280 nm (ε

280

= 7000/M/cm). Add 10 µL to 1 mL of liquid scintillant (Ecoscint H

or equivalent), mix well, and determine the amount of incorporated radioactivity

by liquid scintillation counting. The specific activity of the labeled peptide

(σ

α-MSH

) (in nCi/nmol) is calculated from Eq. 1. The effective specific activity

of the [

H] formaldehyde (σ

[

H] formaldehyde

) is simply half this value (Eq. 2) (see

Note 4):

a-MSH

= (dpm)/(No. nmoles counted × 2220) (1)

[

H] formaldehyde

α-MSH

(2)

3.3. Surface Labeling of Protein

and DNA–Protein Complex by Reductive Methylation

and Quantification of Residues Modified

Prior to mapping the positions of modified lysine residues, it is necessary to

determine the number of residues susceptible to reductive methylation and the

proportion of these that are protected by the presence of DNA.

1. Prepare approx 3.0 mL of a solution of the DNA-binding protein in 10 mM

HEPES, 50 mM NaCl, and 1 mM EDTA (pH 7.5) by either dialysis or buffer

exchange. The concentration of DNA-binding protein in this solution should be

1–2 mg/mL.

2. Take 1.5 mL of protein and add an equimolar amount, or slight excess, of the

DNA duplex from a concentrated stock solution to form the DNA–protein complex.

3. Withdraw a 300-µg aliquot from the free protein and the DNA–protein complex

samples and add glycine to 50 mM from a neutral 1 M stock solution. Dilute the

samples to 1 mL with 50 mM TBS and load into dialysis cassettes (see Note 5).

Dialyze against 2 L of 50 mM TBS at 4°C. These samples are controls for

efficiency of the whole procedure and also serve as the “zero” time points

(see below).

4. To the remaining solutions, add a 30-fold molar excess of sodium cyanoboro-

hydride over the total lysine content of the protein. Then, add a 10-fold molar

excess of [

H] formaldehyde (see Note 6) and incubate at 24°C.

5. At timed intervals (10 min up to 5 h) withdraw 300 µg samples of protein from

each time-course. Quench the reaction by the addition of 50 mM glycine and