Determination of protein oligomerization state: two approaches based on glutaraldehyde crosslinking.
ABSTRACT Many biochemical and biophysical methods can be used to characterize the oligomerization state of proteins. One of the most widely used is glutaraldehyde crosslinking, mainly because of the minimum equipment and reagents required. However, the crosslinking procedures currently in use are impaired by the low specificity of the reagent, which can chemically bond any two amino groups that are close in space. Thus, extensive and time-consuming investigation of the reaction conditions is usually required. Here we describe two approaches based on glutaraldehyde that readily give reliable results.
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ABSTRACT: Two different pyridoxal 5'-phosphate-containing l-threonine deaminases (EC 22.214.171.124), biosynthetic and biodegradative, which catalyze the deamination of l-threonine to alpha-ketobutyrate, are present in Escherichia coli and Salmonella typhimurium. Biodegradative threonine deaminase (TdcB) catalyzes the first reaction in the anaerobic breakdown of l-threonine to propionate. TdcB, unlike the biosynthetic threonine deaminase, is insensitive to l-isoleucine and is activated by AMP. In the present study, TdcB from S. typhimurium was cloned and overexpressed in E. coli. In the presence of AMP or CMP, the recombinant enzyme was converted to the tetrameric form accompanied by significant enzyme activation. To provide insights into ligand-mediated oligomerization and enzyme activation, crystal structures of S. typhimurium TdcB and its complex with CMP were determined. In the native structure, TdcB is in a dimeric form, whereas in the TdcB.CMP complex, it exists in a tetrameric form with 222 symmetry and appears as a dimer of dimers. Tetrameric TdcB binds to four molecules of CMP, two at each of the dimer interfaces. Comparison of the dimer structure in the ligand (CMP)-free and -bound forms suggests that the changes induced by ligand binding at the dimer interface are essential for tetramerization. The differences observed in the tertiary and quaternary structures of TdcB in the absence and presence of CMP appear to account for enzyme activation and increased binding affinity for l-threonine. Comparison of TdcB with related pyridoxal 5'-phosphate-dependent enzymes points to structural and mechanistic similarities.Journal of Biological Chemistry 01/2007; 281(51):39630-41. · 4.65 Impact Factor
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ABSTRACT: The arginine-dependent extreme acid resistance response of Escherichia coli operates by decarboxylating arginine. AdiC, a membrane antiporter, catalyzes arginine influx coupled to efflux of the decarboxylation product agmatine, effectively exporting a proton in each turnover. Using the adiC coding sequence under control of a tetracycline promoter in an E. coli vector, we expressed and purified the transport-protein with a yield of approximately 10 mg/liter bacterial culture. Glutaraldehyde cross-linking experiments indicate that the protein is a homodimer in detergent micelles and lipid membranes. Purified AdiC reconstituted into liposomes exchanges arginine and agmatine in a strictly coupled, electrogenic fashion. Kinetic analysis yields K(m) approximately 80 microm for Arg, in the same range as its dissociation constant determined by isothermal titration calorimetry.Journal of Biological Chemistry 02/2007; 282(1):176-82. · 4.65 Impact Factor
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ABSTRACT: Rabbit and mouse IgG treated with glutaraldehyde (GA) were immunogenic in homologous species. Glutaraldehyde treatment induced in the IgG molecule two types of antigenic determinants. One of them was found on the monomeric fraction of GA-treated rabbit IgG (haptenic determinant) and the other on the polymeric fraction (structural determinant). The haptenic determinants were found also on monoaldehyde-treated rabbit IgG and GA-treated Fab and Fc fragments. It was demonstrated that rabbit and mouse antibodies are specific for GA-treated IgG and have species specificity. While GA treatment did not alter the antigen binding capacity of rabbit IgG antibody, its effector functions (except protein A binding) were much affected. Thus it was found that GA treatment enhances IgG ability to react with rheumatoid factor, reduces drastically its capacity to activate the complement system, abolishes the cytophilic properties of IgG and accelerates its catabolic rate. The possible blocking effect of GA on the amino acid residues (mainly Lys) situated in or very close to the effector sites of the IgG molecule is suggested.Molecular Immunology 08/1983; 20(7):709-18. · 2.65 Impact Factor
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Notes & Tips
Determination of protein oligomerization state: Two approaches
based on glutaraldehyde crosslinking
Vasiliki E. Fadoulogloua,b, Michael Kokkinidisa,c, Nicholas M. Glykosb,*
aDepartment of Biology, University of Crete, PO Box 2208, GR-71409 Heraklion, Crete, Greece
bDepartment of Molecular Biology and Genetics, Democritus University of Thrace, Dimitras 19, 68100 Alexandroupolis, Greece
cInstitute of Molecular Biology and Biotechnology, PO Box 1527, Vasilika Vouton, GR-71110 Heraklion, Crete, Greece
Received 3 September 2007
Available online 24 October 2007
Many biochemical and biophysical methods can be used to characterize the oligomerization state of proteins. One of the most widely
used is glutaraldehyde crosslinking, mainly because of the minimum equipment and reagents required. However, the crosslinking pro-
cedures currently in use are impaired by the low specificity of the reagent, which can chemically bond any two amino groups that are
close in space. Thus, extensive and time-consuming investigation of the reaction conditions is usually required. Here we describe two
approaches based on glutaraldehyde that readily give reliable results.
? 2007 Elsevier Inc. All rights reserved.
Crosslinking by glutaraldehyde is often used to obtain
preliminary information on the quaternary association of
proteins (for instance, see Refs. [1,2]). The simplicity of
the procedure, which requires only the mixing of glutaral-
dehyde with the protein solution [3–5], the availability of
glutaraldehyde as a common reagent easily found in a bio-
chemical laboratory, and the direct detection of crosslinked
products by sodium dodecyl sulfate–polyacrylamide gel
electrophoresis (SDS–PAGE) have led to the wide applica-
tion of this method. However, the results are often ambig-
uous and uninformative. The major limitation of the
technique arises from the nonspecificity of the reagent,
which can react with all the nitrogens of a protein  and
mainly with lysines, tyrosines, histidines, and arginines
[7,8]. Intra- and intermolecular links are formed that could
connect atoms of neighboring but not interacting mole-
cules, yielding artificial protein oligomers that lack biolog-
ical significance. To eliminate the possibility of artificial
interactions, the proper reaction conditions must be estab-
lished through a detailed and often time-consuming investi-
gation of the influence of many parameters such as protein
of reaction . Here we propose two protocols for readily
identifying the oligomerization state of a protein without
an exhaustive examination of reaction conditions. This is
achieved by avoiding the direct mixing of protein with glu-
taraldehyde solution, which is the main deviation from the
conventional ‘‘in-solution’’ approaches. The procedures de-
scribed below expand on previously reported ideas [9,10],
and they have been adapted for investigating the quaternary
association of a protein in native conditions, optimized for
giving rapid and reliable results, and tested to ensure a
‘‘ready-to-use’’ protocol for general application.
The first procedure includes an affinity chromatography
step, is applicable to proteins that carry an affinity tag, and
has been refined and presented here specifically for His-
tagged proteins , which today have wide application.
It has been demonstrated that immobilization of a protein
onto an affinity matrix at low density could be critical to
applications including bifunctional reagents [9,12]. Our
protocol requires amounts of protein as small as 500 lg
and employs a Ni–NTA column (Qiagen). The sample is
diluted to a final volume of 4 ml with 10 mM imidazole,
0003-2697/$ - see front matter ? 2007 Elsevier Inc. All rights reserved.
*Corresponding author. Fax: +30 2551030613.
E-mail address: firstname.lastname@example.org (N.M. Glykos).
Available online at www.sciencedirect.com
Analytical Biochemistry 373 (2008) 404–406
Author's personal copy
50 mM NaCl, 50 mM phosphate buffer, pH 8.0 (buffer A).
It is better to avoid buffers containing amino groups such
as Tris because the reactivity of these groups with glutaral-
dehyde could decrease the yield of the desired reaction (be-
tween protein and glutaraldehyde). A final volume of
0.8 ml of Ni–NTA agarose resin (Qiagen) is packed into
a column (0.8 · 4 cm) and equilibrated with buffer A. The
sample is slowly (at approximately 60 ml/h) applied to
the column. Alternatively, the resin could first bind the
protein in a batch mode and then be packed into a column.
Two washing steps follow: the first consists of 8 ml (10 bed
vol) buffer A, and the second, 8 ml (10 bed vol) of 50 mM
NaCl, 50 mM phosphate buffer, pH 8.0 (buffer B). Glutar-
aldehyde is diluted with buffer B in a final concentration of
0.05% (v/v), and 2.5 ml of this solution (about 3 bed vol) is
added on the column and left to pass through the resin by
gravity. The excess reagent is washed out with 8 ml of
0.5 M Tris/HCl, pH 8.0, which also serves to quench the
reaction. The bound protein is eluted with 300 mM imidaz-
ole, 50 mM NaCl, 50 mM phosphate buffer, pH 8.0, and
the covalent oligomers are detected by SDS–PAGE. All
steps are carried out at room temperature. To eliminate
artificial interactions, that is, the formation of bridges be-
tween different quaternary entities, the protein is spread
out on the matrix in a low protein/matrix ratio (0.6 mg/
ml). Moreover, an even distribution of the protein on the
resin is ensured by the presence of a low concentration of
imidazole in the loading buffer. This requirement might
be better achieved by the alternative procedure, in which
the resin first binds the protein in a batch mode and then
is packed into a column. Fig. 1A and B illustrate applica-
tion of the protocol to the proteins Rop and BcZBP,
respectively. SDS–PAGE analysis of the crosslinked prod-
ucts clearly indicates the dimeric association of the former
and the hexameric association of the latter, results that are
consistent with previous studies [13–15].
The second protocol also requires small amounts of
sample, has wider application than the first protocol, and
is even milder because the protein is not directly mixed with
the glutaraldehyde but the volatility of the reagent is
exploited. A related procedure has been previously de-
scribed for crosslinking of protein crystals for cryocrystal-
lography . The experimental setup includes siliconized
coverslips (microscope coverglasses of 22 mm, Molecular
Dimensions Ltd.), a Linbro-like cell culture plate (XRL
24 well plate, Molecular Dimensions Ltd), vacuum grease
sealant (Dow Corning high-vacuum grease, Hampton Re-
search), microbridges (Hampton Research), and a water
bath for temperature control. The experimental arrange-
ment, which is illustrated in Fig. 2 is similar to a hanging
drop crystallization experiment and proceeds as follows:
A microbridge is filled with 40 ll of 25% (v/v) glutaralde-
hyde acidified with 1 ll of 5 N HCl and is placed on the
bottom of the well. Alternatively, the glutaraldehyde solu-
tion could be placed directly on the bottom of the well
without the microbridge. In that case, the only deviation
from the procedure described below would be the greater
time needed for reagent evaporation and crosslinking.
Ten to fifteen microliters of approximately 0.5 mg/ml pro-
tein solution is placed onto the coverslip which is then used
to seal the well, with the protein solution left to form a
hanging drop inside the well. A small amount of grease ap-
plied to the rim of the well ensures isolation of the system
from the environment. The entire arrangement can be
placed in a water bath for temperature control. Many time
intervals can be checked simultaneously. Application of the
protocol is exemplified for Rop and BcZBP proteins, and
Fig. 1. Glutaraldehyde crosslinking of proteins for the identification of
quaternary protein structure. (A) SDS–PAGE, 15% Laemmli of the His-
tagged Rop protein treated according to the first protocol. Lane 1: Sample
loaded onto the Ni–NTA agarose matrix (the molecular weight of
monomers is approximately 7 kDa). Lanes 2–4: First, second, and third
elution volumes from the column of His-tagged protein after glutaralde-
hyde treatment. A second population of molecular weight <20 kDa has
appeared, clearly indicating the presence of dimers. Lane 5: Low-
molecular-weight marker. (B) SDS–PAGE, 12% Laemmli of the His-
tagged BcZBP protein treated according to the first protocol. Lane 1:
Sample loaded onto the Ni–NTA agarose matrix. Monomers run as a
band with apparent molecular weight of about 30 kDa. Lanes 2–4: first,
second, and third elution volumes from the column of His-tagged protein
after glutaraldehyde treatment. Most of the sample runs as a population
with apparent molecular weight <200 kDa, indicating the presence of
hexamers. Lane 5: High-molecular-weight marker. (C) SDS–PAGE, 15%
Laemmli of the Rop protein treated according to the second protocol.
Lanes 1 and 10: Low-molecular-weight marker. Lane 2: Original sample
not treated with glutaraldehyde. Lanes 3–9: Influence of glutaraldehyde
vapor on the protein solution at different time intervals: 10, 20, 30, 40, 60,
120, and 180 min. A second population of molecular weight corresponding
to that of dimers has appeared, consistent with results obtained from the
first protocol (A). (D) SDS–PAGE, 12% Laemmli of the BcZBP protein
treated according to the second protocol. Lane 1: Original sample not
treated with glutaraldehyde. Lanes 2–6: Influence of glutaraldehyde vapor
on the protein solution for different time intervals: 10, 20, 30, 40, and
60 min. Most of the sample runs as a population with an apparent
molecular weight corresponding to that of hexamers, consistent with
results obtained from application of the first protocol (B). Lane 7: High-
Notes & Tips / Anal. Biochem. 373 (2008) 404–406
Author's personal copy
the results are demonstrated in Fig. 1C and D. SDS–PAGE
analysis of the crosslinked products of Rop (Fig. 1C) re-
veals slow conversion from monomers to dimers. The time
intervals are 10, 20, 30, 40, 60, 120, and 180 min while the
temperature is 30 ?C. SDS–PAGE analysis of the cross-
linked BcZBP protein, which is illustrated in Fig. 1D, re-
veals different behavior. The conversion from monomers
to hexamers is almost completed in the first 20 min of the
experiment. The time intervals are 10, 20, 30, 40, and
60 min and the temperature is maintained at 30 ?C.
In conclusion, we have described two procedures for
glutaraldehyde-based crosslinking of proteins that allow
the efficient and dependable determination of their oligo-
merization state. Our results suggest that these procedures
are generally applicable to all globular proteins.
providing pure protein samples, and members of the ‘‘Vass-
ilis Galanopoulos’’ Electron Microscopy laboratory for
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Hanging drop of the protein solution
Cell culture well Microbridge
Fig. 2. Schematic diagram illustrating the entire experimental setup, used
in the second protocol.
Notes & Tips / Anal. Biochem. 373 (2008) 404–406