Biological Sciences 11 Spring Experiment 4. Protein crosslinking

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1 Biological Sciences 11 Spring 2000 Experiment 4. Protein crosslinking = C - CH 2 - CH 2 - CH 2 - C = H H GA Cl - H 2 N N H 2 Cl - C - CH 2 - CH 2 - CH 2 - CH 2 - CH 2 - CH 2 - C DMS CH 3 CH 3 N - - C - CH 2 - CH 2 - CH 2 - CH 2 - CH 2 - CH 2 - C - - N DSS EGS N - - C - CH 2 - CH 2 - C - - CH 2 - CH 2 - C - - CH 2 - CH 2 - C - - N Bifunctional crosslinker Mol wgt Spacer Arm (Å ) Glutaraldehyde (GA) Dimethylsuberimidate (DMS) Disuccinimidylsuberate (DSS) Ethylene glycolbis[succinimidylsuccinate] (EGS)

2 Crosslinking involves the chemical reaction of a bifunctional reagent with two amino acid residues in the same protein, in adjacent subunits, or in two proteins that associate with each other. The length of the spacer arm between the two functional groups can be adjusted to favor intramolecular crosslinking (short spacer, approx. 5 Å) or intermolecular products (long spacer, typically 11 to 16 Å). The functional groups at each end may be the same (homobifunctional) or different (heterobifunctional). For example, one may use a set of homobifunctional reagents with different spacers to examine the proximity of lysine or arginine residues on subunits of a multi-subunit protein. For measuring the interaction of two different proteins such as a hormone and its cell-surface receptor, one might choose a heterobifunctional reagent with a long spacer arm. The functional groups of the crosslinker reagent are designed to react with amino groups (lys, arg), with sulfhydryls (cys), or with carboxyls (asp,glu), to cite a few examples. Photoreactive moieties such as azides that react non-specifically with nearby molecules have been used as crosslinkers, and special features like water or lipid solubility have been included in the design to allow some preferential reactivity with domains of the biomolecular assembly. Even though many options are available to the biochemist, one of the most frequently used starting points is to test the reactivity of amino groups of the protein with crosslinkers targeted to lys and/or arg. For these residues, the reactive group lies at the end of the residue, somewhat remote from the peptide backbone, and thus it is accessible to extrinsic reagents. The subunit composition of a protein can be determined by crosslinking the subunits of an oligomer and determining the size of the crosslinked product compared to that of the unaltered monomer. We will use amino-reactive reagents with different functional groups and spacer arms to examine the quaternary structure of β-galactosidase, the enzyme studied in Experiments 2 and 3. The products of reaction will be examined by SDS-PAGE on 4-15% polyacrylamide gels. We have the option of running reaction products on 5% gels that facilitate resolution of high molecular weight products. Glutaraldehyde (~ 5 Å spacer) is a classical reagent for crosslinking; it is used in fixation of specimens for microscopy. Since our goal in crosslinking is to examine properties of an oligomeric protein, we will confine its reaction with proteins to short times. This reagent reacts rapidly with amino groups and can effect crosslinking in seconds or minutes. There is speculation that glutaraldehyde reacts preferentially with amino groups not exposed to water and thus is more specific for intrasubunit crosslinking as compared to crosslinking of oligomers to one another.

3 Imidoesters such as dimethyl suberimidate (11 Å) were initially used to to study the quaternary structure of aldolase and several other proteins. The disadvantage of the imido esters is that the reaction is slow (relative to glutaraldehyde) and the competing reaction (hydrolysis) can result in low yields of crosslinked product. At ph 9 (compared with ph 7) reaction of the imidoester with lys and arg is improved. Succinimidyl reagents (DSS and EGS) offer improved yields of crosslinked products. Disuccimidyl suberate and ethylene glycol bis[succinimidylsuccinate] are water-insoluble and must be introduced to the protein solution as a dimethylsulfoxide solution. By using crosslinkers having different reactivity and different spacer arm lengths, it is possible to probe the intramolecular proximity of reactive amino acid residues under selected conditions (such as the presence of substrates or inhibitors) and to probe the intermolecular organization (quaternary structure). As an example, β-galactosidase subunits have been described as having three components to the primary sequence; the first two have similar amino acid composition and share some sequence homology, but the C-terminal third of the protein differs from the N-terminal 2/3 of the protein. The C-terminal portion of the primary sequence contains more lysine residues than does the N-terminal portion. ~ ~ ~ ~ ~ ~ ~~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ Solutions and Reagents Practical Note: DMS, DSS, and EGS are subject to hydrolysis. Therefore we must prepare fresh solutions immediately before use. Hepes buffer, 1 M, ph 9.0 (for dilution of β-galactosidase in 50 mm Hepes) β-galactosidase (1 mg/ml) in storage buffer, Hepes 50 mm, ph 7.5, 50 mm NaCl, 10 mm KCl, 1 mm MgS 4 (dialyzed for DTT removal) Glutaraldehyde (GA) 4% (mw ~ 100; 0.4 M) in water Dimethyl suberimidate (DMS), solid reagent Suggested stock solution is 25 mg/ml in 100 mm Hepes, ph 9.0 Dissolve 12.5 mg in 0.5 ml 100 mm Hepes, and share the solution with other partnerships in your work area. DSS solid. EGS solid. DSS and EGS: Prepare 10 mg/ml stock solutions in dimethylsulfoxide (DMS) in the hood. Dissolve 5 mg in 0.5 ml DMS and share with nearby students. Dimethylsulfoxide (DMS) liquid. [brown bottle in hood]

4 SDS-PAGE Sample Buffer (10X) [supplied in small plastic tubes] 40% glycerol 10% SDS 0.5 M Tris-HCl, ph mM TCEP,HCl 0.01% bromophenol blue SDS-PAGE Running Buffer: (10 X) [in a plastic carboy near the sink] 1% SDS 2.5 M Tris 2.0 M glycine ph 8.3 GelCode Blue Stain Solution (aqueous suspension of Coomassie Brilliant Blue G-250) ~ ~ ~ ~ ~ ~ ~~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ Procedure (a) Since there are 15 lanes on the gel, 13 conditions can be tried. That is Fifteen minus one marker lane and one control lane. The following suggestions are a starting point. We suggest that you calculate molar ratios of [crosslinker]/[protein]. You may alter this protocol to fit your knowledge of relative reactivity of amine derivatization reagents and side reactions. (b) Make a plan of reaction components, and incubation times for 14 tubes. Total volume is 100 µl. Leave room for adding SDS loading buffer at the end. (c) Prepare the Eppendorf tubes with β-galactosidase, buffer, and water; gently vortex mix. Have everything ready before you prepare the crosslinker solution, remembering that loss of active crosslinker from hydrolysis is a competing reaction. (d) Add crosslinker. Vortex mix. Place tubes in rack on bench top. Vortex mix the DMS, DSS and EGS reactions at 10, 5, and 5-min intervals. (e) Terminate the reaction by adding 10 µl SDS loading buffer. Remember to vortex the tube of loading buffer first, since contents tend to settle to the bottom. Glycerol (density=1.26 g/ml) must be uniformly distributed in the loading buffer so that your sample sinks to the bottom of the well when you apply it to the gel.

5 Hepes ( µl ) M C µl β-gal GA 4% DMS DSS EGS Time ( min ) Mix at ( min ) SDS Loading µl Space for notes. (f). Load 15 µl of each sample on the gel. 100 Volts for approx 90 min. /// as your teaching fellow recommends Stop when the pink marker ( 62 kd ) is near the bottom. (g). Rinse gel 4 times with 150 ml distilled water. (1) 30 sec; (2, 3, 4) 4 min each rinse. Stain the gel with GelCode Blue for 30 min /// overnight. (h). Photograph or scan your gel.

6 Results section of the report. 1. Show a photo or scan of your gel, and write a caption. What are the approximate values of Mr for the various bands? Mark the Mr in a legend to the side of the gel. 2. In a table, show the molar ratios of crosslinker to protein used in each reaction, identified by lane number. How many bands appear in each lane of the gel? Suggestion: Expand a table like this to show results of 13 reactions (and control). CL [CL]/[protein] [mol/mol] ReactionTime Products 3. Did some of the crosslinkers give a mixture of intramolecular and intersubunit crosslinked products? Describe the features on your gel that may be associated with intra- vs inter-subunit crosslinks. Discussion section. 1. n the basis of the Mr of the native protein deduced by sedimentation and size exclusion chromatography (Expt 3) and the Mr of the β-galactosidase monomer obtained by SDS PAGE, what is the subunit composition of β-galactosidase? 2. n the basis of the crosslinking experiments (Expt 4), what is the subunit composition of β-galactosidase? 3. What are the advantages and disadvantages of the various crosslinking reagents used? Refer to your table and gel image. How could detection of products be improved? You may find it helpful to write out the reactions that occur with each crosslinker and residues on the protein. Consult organic chemistry texts (optional). Teaching fellows may have additional suggestions for your report, based on results of your lab day.

In-gel digestion of immunoprecipitated proteins separated by SDS-PAGE

In-gel digestion of immunoprecipitated proteins separated by SDS-PAGE (Lamond Lab / April 2008)! Perform all the pipetting steps in a laminar flow hood. We routinely do our digestions in our TC room hoods.