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4 Cross-Linking Protocols for Commonly Used Reagents

4 Cross-Linking Protocols for Commonly Used Reagents

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General Approaches for Chemical Cross-Linking

305  C

 ross-Linking of Porcine Luteinizing Hormone with EDC

to Study a and b Subunit Interactions23

a. Dissolve hormone in deionized water to a concentration of 0.2 mg/mL.

b.Adjust pH to 4.75 with HCl.

c.Dissolve EDC in deionized water and add to the hormone solution to a final concentration

of 0.02 M.

d.Gently stir at room temperature for 1.5 h, keeping the pH constant.

e.Terminate the reaction by dialyzing the reaction mixture against 0.001 N HCl in the cold

and lyophilize for analysis.

9.4.2  Examples for Homobifunctional Reagents  Bis-Imidoesters

Imidates are generally readily soluble in aqueous solutions and are hydrolyzed rapidly with

a pH-dependent half-life ranging from several minutes up to half an hour. Below pH 8.5, the

half-life for ethyl acetimidate is 2–5 min. The rate of hydrolysis increases substantially at higher

pH values.24,25 Up to a 100-fold excess of reagent, at a concentration range of 0.1–10 mM, is

required for complete reaction. To circumvent the degradation problem, incremental additions of

reagents may be used. Imidoesters react over a wide pH range from 7 to 1024–27 and temperatures

from 0°C to 40°C.24,25,28 Alkaline pH increases the rate of the reaction of imidates with amines

to form amidines.24,25,29,30 Imidoester conjugation is usually performed between pH 8.5 and 9.

The reaction rate decreases several fold as the temperature drops from 39°C to 25°C and again

from 25°C to near 0°C.24 At or below zero, amidination occurs at considerably slower rates and

requires longer reaction times of several hours to overnight.28,31,32 The product carries a positive

charge at physiological pH, as does the primary amine it replaces and therefore does not affect

the overall charge of the protein. The final product should be kept at neutral to acidic pH to retard

hydrolysis.  C

 ross-Linking of the Subunits of HIV-1 Reverse Transcriptase

with Dimethylsuberimidate (DMS)33

1. DMS was prepared immediately before use by dissolving 10 mg in 180 μL of ice-cold triethanolamine (TEA)-HCl (0.15 M, pH 8.2). The pH of the DMS solution was readjusted to

8.2 by addition of 20 μL of 1 M NaOH.

2.Cross-linking was initiated by the addition of 2 μL DMS (final concentration 10 mg/mL) to

8 μL of reaction mixture at 4°C. The final reaction mixture (45 mM TEA buffer, pH 8.2)

contained 110 nM heterodimeric HlV-I reverse transcriptase, 650 nM of polyuridylic acid,

10 mM MgCl, 100 mM NaCl, and 1 mM DTT.

3.Incubation was performed for 30 min at 37°C.

4.The cross-linking reaction was stopped by adding an equal volume of 1 M glycine.  Bis-N-Hydroxysuccinimide (NHS) Esters

The NHS cross-linkers are more stable in solution than their imidate counterparts and are typically

more reactive at neutral pH. The half-life of hydrolysis of NHS esters is approximately 10 min at pH

8.6 and 4°C,34 1 h at pH 8.0 and 25°C,35 and 4–5 h at pH 7 at 0°C.36 NHS esters are more stable when

dissolved in dry organic solvents. In absolute ethanol at 23°C, the NHS ester retains 80% activity after 20 days.37 The imidazole group of histidine effectively accelerates the rate of hydrolysis

of the NHS groups in solution. The reaction product of NHS esters with histidine is unstable and

hydrolyzes very rapidly. Therefore, when histidine side chains are present, higher concentration of

NHS cross-linkers are required to achieve a given degree of conjugation. The extent of hydrolysis


Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation

of NHS esters in aqueous solutions may be determined by measuring the increase in absorbance

at 260 nm. The molar extinction coefficient for the NHS group is 8.2 × 103 M−1 cm−1 at 260 nm,

pH 9.0.37 The optimal pH for the cross-linking reaction is pH 9. At pH 7, the reactivity is only half

of that at pH 9. Temperature has little effect on the reaction, allowing it to react efficiently even near

freezing. NHS esters have been used in the concentration range 0.05–9 mM with reaction time from

minutes to hours depending on the conditions used.36,38,39 Buffers that contain primary amines, such

as Tris, or reducing agents should be not be used. Most common buffers are phosphate, bicarbonate/

carbonate, HEPES, and borate at concentrations between 50 and 200 mM. The NHS ester reaction

with amines is typically performed between pH 7.0 and 9.0 at room temperature for 30 min to 2 h.

Reaction times at 4°C should be increased fourfold over room temperature incubation times to give

similar results.22 NHS cross-linkers are usually used in 2- to 50-fold molar excess to protein. The

concentration of the cross-linker can vary from 0.1 to 10 mM. Insoluble NHS esters can be first dissolved in water-miscible organic solvents such as DMSO and DMF. The final organic solvent can

be up to 10% final volume in aqueous reaction. When only the surface of a cell or membrane is to

be modified, it is best to use water-soluble reagents since they will not permeate the membrane. It is

recommended that the protein concentration be kept above 10 μM (50–100 μM) because more dilute

protein solutions result in excessive hydrolysis.  Cross-Linking of Proteins with Ethylene Glycol Bis(Succinimidylsuccinate) (EGS)40

1. The protein of interest has to be taken up in a buffer free of amines, such as HEPES.

The pH of the buffer should be between 7 and 9.

2.Prepare a fresh stock solution of EGS (50 mM) in DMSO. If the protein cannot tolerate

DMSO, choose a different cosolvent or use water soluble sulfo-EGS.

3.Use 10 μg of protein sample per reaction and add EGS to a final concentration of 0.01–5 mM.

4.Incubate the reaction for 20 min at room temperature or on ice for 1 h.

5.Add 50 mM Tris pH 8.0 buffer to quench the reaction; incubate for 5 min at room

temperature.  C

 ross-Linking ATP Synthase Complex with

Dithiobis(Succinimidyl Propionate) (DSP)41

1. Prepare protein sample (1.5 mg/mL) in 50 mM triethanolamine HCl buffer, pH 8.0, containing 0.25 M sucrose.

2.Prepare 0.15 mM DSP stock solution in methanol:acetone (1:1).

3.Incubate protein sample with 1% by volume of DSP at 0°C for 30 min.

4.Quench the cross-linking reaction by addition of lysine to 5 mM final concentration. If

preservation of enzymatic activity is not essential, the reaction can be quenched with

125 mM Tris-HCl buffer, pH6.8, containing 4% SDS, 4 mM EDTA, and 20% glycerol for

gel electrophoresis analysis.

5.Cross-linked proteins can be cleaved by incubating with 80 mM 2-mercaptoethanol or

10 mM DTT.  Bis-Maleimido Reagents

The maleimide group is selective for sulfhydryl groups when the pH of the reaction mixture is kept

between 6.5 and 7.5,42 with an optimum pH for the reaction near 7.0. At pH 7, the rate of the reaction

of maleimides with sulfhydryls is 100-fold faster than with amines. Above this pH range, the reaction rate with primary amines becomes more significant. Above pH 8.0, hydrolysis of maleimides

to nonreactive maleamic acid can occur.43 Reducing agents should be excluded from buffers, since

they will quench the reactivity. β-Mercaptoethanol, dithiothreitol, mercaptoethylamine, and other

thiol compounds must be removed prior to the cross-linking reaction.

General Approaches for Chemical Cross-Linking

307  C

 ross-Linking of Cysteine-Containing Mutant of Mitochondrial

F1Fo ATP Synthase Complex with 1,6-Bis-Maleimidohexane (BMH)

or N,N′-o-Phenylene-Dimaleimide (OPD)44

1. Stock solutions (100 mM) of BMH and OPD in dimethylformamide were stored at −20°C.

2.Mitochondria with cysteine-containing mutant of F1Fo ATP synthase complex were prepared and suspended in the isolation buffer (0.6 M mannitol, 2 mM EGTA, 10 mM Trismaleate, pH 6.8).

3.These prepared mitochondria were washed twice with 0.6 M mannitol, 2 mM EGTA,

50 mM HEPES, pH 7.0, and suspended in the same buffer at a protein concentration of

5 mg/mL.

4.This suspension was incubated with 300 μM of either OPD or BMH for 1 h at room


5.Reactions were stopped by the addition of 25 mM of 2-mercaptoethanol.  Bis-α-Haloacetyl Reagents

Haloacetyl compounds are directed toward the sulfhydryl but also react with other functional

groups. The most common reagents are α-iodoacetyl derivatives. Selectivity for sulfhydryl groups is

achievable by using only a slight excess of γ-haloacetyl groups over the number of free sulfhydryls

and by keeping the pH of the reaction mixture between 7.5 and 8.5, with an optimum specificity at

pH 8.3. If there are no free sulfhydryls present, or a gross excess of haloacetyl group is used, the

haloacetyl group can react with other amino acids such as imidazoles at pH 6.9–7.0, although the

reaction is slow. Extraneous reducing agents should be excluded from buffers for α-haloacetyl reactions. Iodoacetamides commonly used to modify thiols will react with amines of proteins if the pH

is in the range 9.0–9.5.45  C

 ross-Linking of Aldolase Subunits with

N,N-Bis(α-Iodoacetyl)-2,2′-Dithiobis(Ethylamine) (BIDBE)46

1. Prepare 7.15 mg/mL aldolase solution in 0.05 M Tris-HCl buffer, pH 8.0.

2.Prepare a 34.1 mM solution of BIDBE, freshly dissolved in dimethylsulfoxide.

3.0.25 mL of aldolase was mixed with 10 μL of BIDBE. The final concentration of BIDBE is

1.31 mM and the molar ratio of BIDBE to aldolase sulfhydryls is 0.95:1.

4.Incubate the mixture for 3.5 h at 25°C in the dark.

5.At the end of the incubation, the sample is dialyzed for 16 h at 1°C in the dark against

0.059 M Tris-phosphate buffer, pH 7.0, containing 20% sucrose.

6.The sample is then analyzed by gel electrophoresis.

7.The cross-linked protein can be cleaved by 1% β-mercaptoethanol.

9.4.3  Examples for Heterobifunctional Reagents

Heterobifunctional reagents are diverse compounds as discussed in Chapter 6. The reaction conditions described above for specific reactive moieties also apply to the heterobifunctional crosslinkers if they contain such a reactive group. In general, the most reactive or unstable moiety end

of the bifunctional agent is reacted first. For example, with NHS-ester-disulfide cross-linkers such

as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) reaction with an amine is carried out first

to react with the NHS ester. The pyridyl–disulfide derivative obtained can either be reduced with

reducing agents such as β-mercaptoethanol to generate a free thiol or reacted directly with protein

or peptide with a free sulfhydryl for coupling via disulfide exchange. The pyridine-2-thione released

can be quantified at 343 nm (molar extinction coefficient at 343 nm = 8.08 × 103 M−1 cm−1).47,48 For

N-succinimidyl S-acetylthioacetate (SATA) after reaction with an amine, excess and hydrolyzed


Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation

SATA is typically removed by gel filtration or other similar techniques. The activated protein is

usually quite stable and can be stored for long periods. The blocked sulfhydryl group on the derivatized molecule can be deprotected by incubation with 50 mM hydroxylamine hydrochloride for

2 h at pH 7.5. It is not necessary to remove the hydroxylamine from the deprotected derivative prior

to reaction with maleimide cross-linkers. Similarly, for NHS-ester-maleimide compounds, reaction

with NHS ester is first carried out between pH 6.5 and 7.5 to prevent hydrolysis and reaction of the

maleimide group with amines. If it is necessary to perform the maleimide reaction prior to coupling

with the NHS ester, a buffer with pH below 7.0 should be used and the reaction times should be kept

to a minimum. This procedure will be most successful if there are no primary amines available

and if a sulfhydryl is readily accessible. An example is sulfosuccinimidyl-4-(N-maleimidomethyl)

cyclohexane-1-carboxylate (sulfo-SMCC), which is very stable in solution exhibiting essentially no

degradation after 6 h at 30°C in pH 7 buffer. A SMCC activated protein is stable and often can be

stored several months prior to the second stage of coupling.47  C

 onjugation of Human Serum Albumin (HSA) and Monoclonal

Antibody (mAb) with SPDP49

a. One mL of mAb Dal K20 (10 mg/mL) in PBS (0.01 M phosphate in 0.15 M sodium chloride, pH 7.2) was derivatized with fivefold molar excess of SPDP for 30 min. SPDP was

dissolved in a small volume of DMF, which did not exceed 20% of the volume of the HSA

solution. The solutions were then desalted into buffer B (0.1 M sodium phosphate, pH 7.2,

containing 1 mM EDTA (ethylenediaminetetraacetic acid) ). The number of pyridyldithio

groups incorporated into mAb Dal K20 was determined in the presence of 0.1 M DTT,

using E = 8.08 × 103 M−l cm−l at 343 nm for released pyridine-2-thione.

b.To HSA at 10 mg/mL in PBS was added a fivefold molar excess of SPDP with stirring

at room temperature. The SPDP had been dissolved in an amount of DMF that did not

exceed 20% of the volume of the HSA solution. After 30 min, the solution was desalted

into 0.1 M acetate buffer, pH 4.5, containing 0.1 M NaCl, and DTT added to give a concentration of 10 mM. After 20 min at room temperature, the DTT-treated mixture was

desalted into buffer B. The number of pyridyldithio groups incorporated into HSA was


c.mAb Dal K20 (3.6 mg/mL, 3.0 mL) derivatized with SPDP in buffer B was mixed with

HSA-SPDP-SH in buffer B at a 4:1 molar ratio of HSA over mAb Dal K20 in a final volume of 6.2 mL. All reaction mixtures were stirred briefly and left at room temperature

for 16 h at which time thiols were blocked by the addition of a 20-fold molar excess of

N-ethylmaleimide over mAb Dal K20.

d.The mAb K20-HSA conjugate was purified by gel filtration chromatography on Bio-Gel P

300 (2.5 cm × 90 cm) equilibrated with PBS.  C

 ross-Linking of Demineralized Bone Matrix (DBM)

and Monoclonal Antibody with Sulfo-SMCC50

a. Traut’s Reagent (5 mg/mL) was dissolved in PBS with 4 mM EDTA (pH = 8).

b.DBM (5 mm × 5 mm × 2 mm) was soaked in Traut’s Reagent for 3 h at room temperature.

c.In a separate reaction, 5 μg monoclonal antibody was diluted in PBS containing 4 mM

EDTA (pH = 7.2), and sulfo-SMCC (25 μg/mL) was reacted with antibodies at room temperature for 30 min.

d.The DBM treated by Traut’s Reagent was washed by PBS for several times, and incubated

with antibodies treated by Sulfo-SMCC for 2 h at room temperature.

e.The antibody-conjugated DBM was then washed with PBS for several times and incubated

with 5% (W/V) glycine and 5% (W/V) bovine serum albumin for 3 h to block remaining

reactive groups on DBM.

General Approaches for Chemical Cross-Linking


9.4.4  Examples for Heterobifunctional Photosensitive Reagents

Photosensitive reagents are, of course, sensitive to light and they are particularly sensitive to ultraviolet radiation, that is, wavelengths less than 300 nm. Consequently, they must usually be handled in

the dark or under conditions of dim or red light. For example, under normal white fluorescent lamp

illumination, some photoreactive reagents may have half-lives of only a few hours or less. Azides

are also sensitive to sulfhydryl reducing agents such as dithiothreitol, 2-mercaptoethanol, and glutathione, since they can be reduced to the corresponding amine.51,52 This reduction is pH dependent.

In 10 mM dithiothreitol, various azides are found to have half-lives of 5–15 min at pH 8. At pH 10,

this rate is increased 12-fold.52 With 50 mM glutathione or 2-mercaptoethanol at pH 8, the azides

were reduced 60%–70% and 10%–20%, respectively, in 24 h. Therefore, if reducing conditions are

required during cross-linking, the use of 2-mercaptopethanaol is recommended.

Aryl azides are generally insoluble in aqueous buffers and may be solubilized with the aid of

water-miscible organic solvents such as acetone, methanol, ethanol, dioxane, dimethylformamide,

pyridine, acetonitrile and dimethylsulfoxide.39,53–56 The final concentration of organic solvent may

be as high as 20%. Alternatively, a fine powder of the reagent may be added to the reaction mixture,

although the rate of reaction may be reduced.

The photolysis of photosensitive cross-linkers and their subsequent chemical reactions are temperature independent. A common method to photoactivate azides is to irradiate with a short-wavelength UV lamp (typically 254 nm). Arylazides are photolyzed at wavelengths between 250 and

460 nm forming a reactive aryl nitrene. The half-time of photolysis is usually on the order of 10–50 s

with the sample positioned close (e.g., 1 cm) to the lamp.57 An alternative method is flash photolysis

using electronic flash units.32,58,59 A bright camera flash works well with the nitro and hydroxylsubstituted aryl azides. Unsubstituted aryl azides require UV light or numerous flashes. In a typical

experiment, less than 10 flashes are sufficient to photolyze reagents associated with proteins.59 With

molecules that contain chemical moieties that are photosensitive or prone to photo-oxidation, longer

wavelength illumination may be necessary, for example, by illumination through glass filters which

are particularly useful for activating aryl azides with nitro substituents.60 In this case, irradiation

must be lengthened to many minutes or even hours, depending on the molar absorptivity of the aryl

azides at the longest wavelengths.39,61,62 It should be kept in mind that the yield resulting from a photoreactive cross-linker is inherently low. Yields of less than 10% should be considered acceptable.47  C

 ross-Linking of Proteins with the Photoreagent N-(4-Azido-2,3,5,

6-Tetrafluorobenzyl)-3-Maleimidylpropionamide (TFPAM-3)56  Labeling

1. Pass protein through a BioSpin 6 centrifuge column equilibrated with 50 mM Mops buffer (pH 7.0), containing 0.5 mM EDTA, and 10% glycerol to remove thiols and primary

amines present in storage buffer.

2. Prepare a stock solution of 4 mM TFPAM-3 in DMSO.

3. Mix 40 μM of a protein with 250 μM TFPAM-3 at room temperature for 1 h with gentle stirring.

4. Quench the reaction with 1 mM cysteine for 30 min at room temperature with gentle stirring.

5.Remove excess TFPAM-3 by passing the sample through a column equilibrated with

25 mM Hepes (pH 7.5), containing 60 mM potassium acetate, 6 mM magnesium acetate,

and 10% glycerol (conjugating buffer). Proteins can be stored in this buffer at 20°C.  Photo-Conjugation

1. Mix 1 μM of TFPAM-3 labeled protein with 1 μM of a protein of interest in conjugating


2. Irradiate the sample for 45 min at room temperature at a distance of about 2 cm from the

UV lamp (6W UV lamp, model UVL-56, Blak-Ray lamp).

3.Analyze conjugated products.


Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation  C

 ross-Linking UvsY Hexamer Protein Complex with the Photo-Reagent

Ruthenium(II) Tris-Bipyridyl Dichloride (Ru(II)bpy3Cl2)63

a. Free UvsY protein from DTT and β-mercaptoethanol as they can be oxidized in the


b.Mix proteins (0.01–20 μM in 15 mM sodium phosphate (pH 7.5) containing 150 mM NaCl)

with 0.125 mM Ru(II)bpy3Cl2.

c.Place the solution in a 1.7 mL Eppendorf tube parallel to the beam of light at a distance of

50 cm from a 150-W xenon arc lamp, or at a distance of 5 cm for the high-intensity standard


d.Add ammonium persulfate to 2.5 mM concentration just before irradiation.

e.Irradiate sample for 0.5 s with xenon arc lamp (the light should be filtered first through

10 cm of distilled water and then through a 380–2500 nm cut-on filter. Exposure time can

be controlled by shining light through the shutters of a single lens reflex camera with the

lens and back cover removed) or for 5–30 s for flashlight.

f.Quench the reaction with approximately 10 μL of 0.2 M Tris, 2.88 M β-mercaptoethanol.

g.Analyze the cross-linked products.


The above discussion on reaction protocols is based on the chemical properties of the cross-linking

reagents. While understanding the chemistry of the reagents is important, in most cases, we would

be most interested in the biological system under study. Based on the aim in studying the biological macromolecules, a specific cross-linker will be chosen. For example, to study a biological

transmembrane system, a reagent that can penetrate the membrane would be desired. Thus, more

hydrophobic and less hydrophilic cross-linkers should be chosen. The reverse would be true for

studying aqueous macromolecules. The following will demonstrate the principles in choosing the

right reagents for some general biological systems to be investigated.

9.5.1  Soluble Macromolecules

Water-soluble macromolecules are those entities that exist freely in aqueous solutions. Most of the

water-soluble proteins have a shell of hydrophilic amino acids such as serine, aspartic acid, and

lysine that can form hydrogen bonds with surrounding water molecules. The core of the soluble protein may contain hydrophobic amino acids such as leucine and tryptophan. Some macromolecules

tend to associate into higher aggregates when present in high concentrations, depending on their

dissociation constants. On the other hand, some macromolecules exist in stable multisubunit entities.

Under certain circumstances, different molecules may associate to form complexes, such as ligand–

receptor interactions. An intriguing example is the interaction between α-lactalbumin and galactosyltransferase. It is only in the presence of glucose and UDP-galactose that the two proteins associate

to form lactose synthase.64 Thus, for studying protein–protein interactions in such systems, appropriate conditions must be chosen for the interaction to occur. Since there are so many different possible

variations of soluble proteins to be studied, only a few examples are chosen to illustrate the principles

involved in the cross-linking process. In general, for cross-linking of soluble macromolecules, watersoluble reagents would be used. As discussed in Chapter 4, compounds with ether-oxygen, hydroxyl

groups, ester and amide bonds, formal charges, and sulfonation have increased solubility in aqueous

solutions. These reagents should be considered first when designing a cross-linking experiment.  Cross-Linking Nonassociated Proteins

Intramolecular cross-linking of nonassociated proteins can be carried out with a one-step procedure. As discussed above in Section 9.2.1, the cross-linker can be added directly to a solution of

General Approaches for Chemical Cross-Linking


target proteins. Since intermolecular cross-linking would also occur, dilute solutions should be used

to avoid close encounter of the macromolecules. For intermolecular conjugation of two proteins,

such as the preparations of immunotoxins or enzyme-antibody conjugates, a two-step procedure is

desired as discussed above under Section 9.2.2, with examples presented therein. Three-step and

multistep procedures are also applicable as shown in Sections 9.2.3 and 9.2.4.  Cross-Linking Multisubunit Complexes

Similar to single subunit proteins, dilute solutions of strongly associated, stable multisubunit complexes should be used to avoid intermolecular cross-linking as discussed in Section 9.3.3. In most

cases, a one-step procedure can be used for intracomplex subunit cross-linking as showed above in

Section 9.2.1 for ribosome protein complexes. Various reagents, either homofunctional or heterofunctional, may be used depending on the aims of the experiments. These are illustrated in Section

9.4 for reverse transcriptase (Section, ATP synthase (Sections and,

aldolase (Section, and UvsY protein hexamer (Section

Investigation of protein–protein interactions of readily dissociable protein complexes is more challenging. Some proteins interact with several other proteins transiently for regulatory or enzymatic

purposes. In these cases, a condition that causes the formation of the complex must be achieved during the cross-linking experiment, either by inclusion of substrates or cofactors that induce the interaction or by increasing the concentrations of the interacting subunits. The most commonly used reagents

are photoactivatable cross-linkers, although oxidative cross-linking such as hexahistidine-mediated

cross-linking methodology has been evolving.65,66 Any of the photoactivatable reagents listed in

Appendix E may be used. In the first step, the reagent is covalently attached to lysine or cysteine side

chains of the prey protein. The derivatized prey protein is then incubated with an interacting protein

and exposed to UV light to induce cross-linking between the prey protein and the interacting proteins.

For label transfer experiments, cleavable reagents, particularly those containing disulfide bonds, are

used. After photoactivation, the reagent is cleaved, thereby transferring the label to the interacting

proteins. The transferred label enables the identification or isolation of the interacting proteins.

For hexahistidine-cross-linking, a hexahistidine tag or a NH2-Gly-Gly-His tag is first genetically

introduced into the prey protein.66–68 On incubation of the tagged prey protein with other interacting

protein in the presence of nickel, fast and efficient cross-linking of proteins is achieved when oxidized by peracids like magnesium monoperoxyphthalic acid or KHSO5. The cross-linked adducts

can be identified immunologically. Klein et al.69 have used this method to study the transcriptional

activation domain–coactivator complexes.

Advances in genetic cloning have facilitated the development of a new methodology of

cross-linking to study protein–protein interaction, particularly in living cells. Photoactivatable

amino acid analogues such as p-benzoyl-l-phenylalanine (pBpa), p-azido-l-phenylalanine, and

4′-[3-(trifluoromethyl)-3H-diazirin-3-yl]-l-phenylalanine are site-specifically incorporated into

interested proteins by means of heterologous amber suppressor tRNA/aminoacyl-tRNA synthetase

pairs that recognize the unnatural amino acids.70 After association of the modified protein with target macromolecules, the complex is photoactivated to initiate cross-linking. Lee et al.71 have used

pBpa to study the interaction of Escherichia coli catabolite activator protein (CAP) with DNA. pBpa

was genetically incorporated into CAP in bacteria in response to an amber nonsense codon using

an orthogonal tRNA/aminoacyl-tRNA synthetase pair. On binding with DNA and after UV irradiation, SDS–PAGE analysis showed that the mutant CAP containing pBpa formed a covalent complex

with a DNA fragment containing the consensus operator sequence.

9.5.2  Membrane-Bound Proteins

Membrane-bound proteins present a special challenge for studying protein–protein interactions.

These proteins consist of transmembrane helices that are hydrophobic in nature and an extramembraneous region exposed to the aqueous media. The choice of cross-linking reagent depends on

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4 Cross-Linking Protocols for Commonly Used Reagents

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