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.
- [Show abstract] [Hide abstract]
ABSTRACT: The structure of BC0361, a polysaccharide deacetylase from Bacillus cereus, has been determined using an unconventional molecular-replacement procedure. Tens of putative models of the C-terminal domain of the protein were constructed using a multitude of homology-modelling algorithms, and these were tested for the presence of signal in molecular-replacement calculations. Of these, only the model calculated by the SAM-T08 server gave a consistent and convincing solution, but the resulting model was too inaccurate to allow phase determination to proceed to completion. The application of slow-cooling torsion-angle simulated annealing (started from a very high temperature) drastically improved this initial model to the point of allowing phasing through cycles of model building and refinement to be initiated. The structure of the protein is presented with emphasis on the presence of a C(α)-modified proline at its active site, which was modelled as an α-hydroxy-L-proline.Acta Crystallographica Section D Biological Crystallography 02/2013; 69(Pt 2):276-83. · 12.67 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Glutaraldehyde (GA) reacts with amino groups in proteins, forming intermolecular cross-links that, at sufficiently high protein concentration, can transform a protein solution into a gel. Although GA has been used as a cross-linking reagent for decades, neither the cross-linking chemistry nor the microstructure of the resulting protein gel have been clearly established. Here we use small-angle X-ray scattering (SAXS) to characterise the microstructure and structural kinetics of gels formed by cross-linking of pancreatic trypsin inhibitor, myoglobin or intestinal fatty acid-binding protein. By comparing the scattering from gels and dilute solutions, we extract the structure factor and the pair correlation function of the gels. The protein gels are spatially heterogeneous, with dense clusters linked by sparse networks. Within the clusters, adjacent protein molecules are almost in contact, but the protein concentration in the cluster is much lower than in a crystal. At the ∼1 nm SAXS resolution, the native protein structure is unaffected by cross-linking. The cluster radius is in the range 10-50 nm, with the cluster size determined mainly by the availability of lysine amino groups on the protein surface. The development of structure in the gel, on time scales from minutes to hours, appears to obey first-order kinetics. Cross-linking is slower at acidic pH, where the population of amino groups in the reactive deprotonated form is low. These results support the use of cross-linked protein gels in NMR studies of protein dynamics and for modeling NMR relaxation in biological tissue.Physical Chemistry Chemical Physics 01/2014; 16(9):4002-11. · 4.20 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The reversible reaction catalyzed by serine hydroxymethyltransferase (SHMT) is the major one-carbon unit source for essential metabolic processes. The Arabidopsis thaliana genome encodes seven SHMT isozymes localized in mitochondria, plastids, nuclei, and the cytosol. Knowledge of the biochemical properties of each isozyme is central to understanding and manipulating one-carbon metabolism in plants. We heterologously expressed and purified three recombinant SHMTs from A. thaliana (AtSHMTs) putatively localized in mitochondria (two) and the cytosol (one). Their biochemical properties were characterized with respect to the impact of folate polyglutamylation on substrate saturation kinetics. The two mitochondrial AtSHMTs, but not the cytosolic one, had increased turnover rates at higher (>0.4 ng/μL) enzyme concentrations in the presence of monoglutamylated folate substrates, but not in the presence of pentaglutamylated folate substrates. We found no experimental support for a change in oligomerization state over the range of enzyme concentration studied. Modeling of the enzyme structures presented features that may explain the activity differences between the mitochondrial and cytosolic isozymes.Archives of Biochemistry and Biophysics 06/2013; · 3.37 Impact Factor
This article was published in an Elsevier journal. The attached copy
is furnished to the author for non-commercial research and
education use, including for instruction at the author’s institution,
sharing with colleagues and providing to institution administration.
Other uses, including reproduction and distribution, or selling or
licensing copies, or posting to personal, institutional or third party
websites are prohibited.
In most cases authors are permitted to post their version of the
article (e.g. in Word or Tex form) to their personal website or
institutional repository. Authors requiring further information
regarding Elsevier’s archiving and manuscript policies are
encouraged to visit:
Author's personal copy
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
technical assistance. V.E.F. is supported by a Postdoctoral
Research Fellowship from the Greek State Scholarships’
 D.K. Simanshu, H.S. Savithri, M.R.N. Murthy, Crystal structures of
Salmonella typhimurium biodegradative threonine deaminase and its
complex with CMP provide structural insights into ligand-induced
oligomerization and enzyme activation, J. Biol. Chem. 281 (2006)
 Y. Fang, L. Kolmakova-Partensky, C. Miller, A bacterial arginine–
agmatine exchange transporter involved in extreme acid resistance, J.
Biol. Chem. 282 (2007) 176–182.
 D. Onica, G. Mota, A. Calugaru, M. Manciulea, S. Dima,
Immunogenicity and effector functions of glutaraldehyde-treated
rabbit and mouse immunoglobulin, G. Mol. Immunol. 20 (1983)
 D.B. McIntosh, D.C. Ross, Role of phospholipids and protein–
protein associations in activation and stabilization of soluble Ca2+-
ATPase of sarcoplasmic reticulum, Biochemistry 24 (1985) 1244–
 M. Kapoor, M.D. O’Brien, Investigation of the quaternary structure
of Neurospora pyruvate kinase by cross-linking with bifunctional
reagents: the effect of substrates and allosteric ligands, Can. J.
Biochem. 55 (1977) 43–49.
 J.A. Kiernan, Formaldehyde, formalin, paraformaldehyde and glu-
taraldehyde: what they are and what they do, Microscopy Today 00-1
 R.L. Lundblad, C.M. Noyes, in: Chemical Reagents for Protein
Modification, CRC Press, Boca Raton, FL, 1984, pp. 123–139.
 J.W. Payne, Polymerization of proteins with glutaraldehyde, Bio-
chem. J. 135 (1973) 867–873.
 S. Pillai, B.K. Bachhawat, Affinity immobilization and ‘‘negative’’
crosslinking: a probe for tertiary and quaternary protein structure, J.
Mol. Biol. 131 (1979) 877–881.
 C.J. Lusty, A gentle vapor-diffusion technique for cross-linking of
protein crystals for cryocrystallography, J. Appl. Crystallogr. 32
 R. Janknedht, G. Martynoff, J. Lou, R. Hipskind, A. Nordheim,
H.G. Stunnenberg, Rapid and efficient purification of native histi-
dine-tagged protein expressed by recombinant vaccinia virus, Proc.
Natl. Acad. Sci. USA 88 (1991) 8972–8976.
 S. Pillai, B.K. Bachhawat, Protein–protein conjugation on a lectin
matrix, Biochem. Biophys. Res. Commun. 75 (1977) 240–245.
 D.W. Banner, M. Kokkinidis, D. Tsernoglou, Structure of the
ColE1 rop protein at 1.7 A˚resolution, J. Mol. Biol. 196 (1987)
 V.E. Fadouloglou, A. Deli, N.M. Glykos, E. Psylinakis, V. Bouriotis,
M. Kokkinidis, Crystal structure of the BcZBP, a zinc-binding
protein from Bacillus cereus: functional insights from structural data,
FEBS J. 274 (2007) 3044–3054.
 V.E. Fadouloglou, D. Kotsifaki, A.D. Gazi, G. Fellas, C. Meramve-
liotaki, A. Deli, E. Psylinakis, V. Bouriotis, M. Kokkinidis, Purifi-
cation, crystallization and preliminary characterization of a putative
LmbE-like deacetylase from Bacillus cereus, Acta Crystallogr. F 62
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