1 52s Biochemical Society Transactlons ( 1 996) 24
FTIR spectroscopic structural analysis of the CHIP28
water channel protein
PARVEZ I. HAMS. DENNIS CHAPMAN & GHEORGHE BENGA"
Dept. of Protein 2 Molecular Biology. Royal Free Hospital School
of Medicine, Rowland Hill St., London, NW3 2PF, UK, "Dept. of
Cell and Mol. Biology, "Iuliu Hatieganu" University of Medicine and
Pharmacy, Cluj-Napoca, 6 Pasteur St., 3400 Cluj-Napoca, Romania
A prominent feature of the red blood cell (RBC) membrane
is its high permeability to water [for a review see reference I].
Consistent with this hypothesis the first channel-forming integral
membrane protein of M,=28 kDa (called CHIP28 or aquaporin) from
RBC was identified by Agre and coworkers (21. The CHIP-mediated
water channel activity was demonstrated both with a Xenopus luevis
oocyte expression system and also with CHIP28 reconstituted into
liposomes [3.4]. In both systems CHIP28 markedly increases the
water permeability, the Arrhenius activation energy is low and
mercurials drastically inhibit the permeability. These model studies
indicate that CHIP28 behaves similarly to the water channels of
native membranes. A detailed knowledge ofthe molecule is however
required in order to further understand the structure-function
relationships of this water channel protein. Fourier-transform infrared
spectroscopy (FTIR) is one of the few techniques which can be
applied to obtain structural information on membrane bound proteins
IS]. This technique was used to investigate the secondary structure
and hydrogen-deuterium exchange properties of the CHIP28 protein
from human red blood cells.
The preparation of purified CHIP28 vesicles used in this
study was adapted from the method of Denker et al. [ 2 ] . The FTlR
second-derivative spectrum of the CHIP28 protein in H,O buffer
shows the major amide I component at 1657cm-' which is attributed
to a combination of a-helical and random coil structures [ 5 ] .
The presence of substantial 0-sheet structure in the protein is
indicated by the band at 1638cm" [ 5 ] .The results of the secondary
structure analysis of CHIP28 reveals virtually equal proportions of ahelical (36%) and p-sheet (42%) structures. This is in good
agreement with the results o f a recent CD analysis of van Hoek et al.
161 which give values of 40% a-helix and 42% P-sheet. There
are some similarities and also differences in the secondary structure
analysis obtained from our FTIR study compared with that obtained
by van Hoek et al. [ 6 ] in their FTIR analysis. In both studies the
content of helical and turn structures are similar. However. the psheet content estimated by van Hoek et al. is only 18%. whereas in
our study we obtain a value of42%. Our result is in good agreement
with their CD value [6]. The FTIR analysis of van Hoek et al. [6]
was carried out for dehydrated samples whereas our analysis was
carried out for samples in an aqueous suspension.
Upon suspension of the CHIP28 protein in 'HzO media,
remarkable spectral changes occur. The amide I band shifts to
1625cm~l. The amide I1 band at 1550cm.' undergoes a large
reduction in intensity. Both this and the large shift of the amide I
band are attributed to 'H-'H exchange of the amide protons [5].
Isotopic substitution of the peptide bond N-H to N-'H results in the
shift of the amide I1 band (1 550cm-I) to lower frequency by 1 OOcm-'
[5]. This can be used for investigation of hydrogen-deuterium
exchange in proteins. The unusually high hydrogen-deuterium
exchange of the CHIP28 protein is illustrated in Figure 1 . This
represents the FTIR spectra of a film dried from an H,O suspension
of CHIP28 protein in membrane vesicles. Addition of some 'H,O to
the dry film results in an immense reduction in the intensity of the
amide I1 band (see Figure 1). Furthermore, the amide I band shifts
by over 20cm-' towards lower frequency. These changes are
observed rapidly (within 5 minutes) after exposure of the protein to
*H,O. Such an unusually high extent of hydrogen-deuterium
exchange (over 80% exchange within five minutes) is rarely
observed with membrane proteins. Due to their membrane
embedded nature most membrane proteins do not display high
hydrogen-deuterium exchange rates.
Figure I : FTlR spectra of the CHIP28 protein i n a partially dehydrated
film (continuous line) and after exposure ofthe film to 'tI,O (brohell line).
Only the glucose transporter protein has been reported to have an
unusually high rate of hydrogen-deuterium exchange 171. This was
interpreted to suggest the possible existence of an aqueous pore in
this protein [ 71. The same conclusion can be reached tor the C'lIlP28
protein. However. the magnitude of the spectral changes in the
amide I band is much greater for CHIP28 than observed with the
glucose transporter. For example. in the absorbance spectrum of
glucose transporter the amide I shifts by only 3cm I in 'HIO.
Whereas for CHIP28 a shift of 20cni.' is observed. These changes
can be interpreted as being due to the polypeptide segments. lining
the channel pore. in the CHIP28 protein being more solvent exposed.
This is likely for a protein that has been proposed to function as a
water channel.
To conclude. the most interesting observation of this study is
the unusually high hydrogen-deuterium exchange of the ClHlP28
protein, a characteristic that tits well with its proposed functional role
as a water channel.
We thank the IRC in Biomedical Materials (PI11 & I)(') and
the Wellcome Trust ( G 1 l . H ) for financial support
[ I ] Benga. Gh. (1988) Prog. Biophys. Mol. Biol. 51. 193.
[2] Denker. B.M., Smith, B.L.. Kuhajda. F.P.. Agre. P. (1988) J .
Biol. Chem. 263. 15634.
[3] Preston. G.M., Carroll. T.P.. Guggino, W.R.. Agre. I]. (1992)
Science, 256. 385.
[4] Zeidel, M.L., Nielsen, S . . Smith, B.L. Ambudker, S.V..
Manusbach. A.B., Agre. P. (1994) Biochemistry 33. 1606.
[ 5 ] Haris, P.I., Chapman. D. (1992) Trends. Biochem. Sci. 17. 32X.
[6]Van Hoek, A.N., Wiener, M.. Bicknese. S.. Miercke, I... Biwersi,
J., Verkman, ,A.S. ( I 993) Biochemistry 32, 1 1847.
[7] Alvarez, J., Lee, D.C.. Baldwin, S.A. and Chapman, D. (1987) J.
Biol. Chem. 262, 3502.