IndianaCTSI ACCELERATING CLINICAL AND

TRANSLATIONAL RESEARCH Indiana Clinical and Translational Sciences Institute

www.indianactsi.org

Measurements of Interaction Between Glycoproteins and

Lipid Bilayers
Kwame E. Newton , Andrew Fraser , Bruce D. Ray , Horia I. Petrache 1 2 2 2

1
University High School of Indiana, 2Department of Physics, IUPUI, Indianapolis, IN 46202

Purpose Results
Cell membranes are mainly composed of lipids and proteins, which
interact with each other in various ways. Glycoproteins, or proteins
with attached sugars, sometimes behave differently than their non- Microenvironment Shifts in the Presence of DOPS
glycosylated counterparts. Some lipids have the ability to alter the
tertiary (three-dimensional) structure of glycoproteins; investigating DOPS DPhPS DOPC DPhPC
these lipid-protein interactions could provide more insight into how Ion Channel
glycoproteins and cell membranes function. Protein Phe Tyr Trp Phe Tyr Trp Phe Tyr Trp Phe Tyr Trp Currentc
Ovomucoid -0.290 0.190 0.580 0.780 -0.340 0.970 0.100 -0.100 -0.030 -0.990 0.900 0.620 0 pAb
Ovalbumin A1a 0.560 0.370 0.917 -0.080 0.290 -1.370 0.600 -0.480 0.540 -1.950 0.020 0.230 100-200 pAb
Methods Ovalbumin A2a -1.260 0.340 0.053 0.460 0.900 0.140 -0.010 -0.310 -0.100 -0.050 0.060 0.580 10-20 pAb
Sample Preparation Lipids were placed in an aqueous solution at Ovalbumin A3a -0.040 -0.140 -0.100 -0.630 0.130 -0.380 0.070 0.270 -0.100 -0.110 0.730 0.170 300-400 pAb
1.2 mg/mL, and a Na/KPO4 pH 8 buffer was added at 5 mM. The Ovoinhibitor 0.240 0.390 -0.340 -0.340 0.300 -0.030 0.190 -0.090 0.050 -0.520 0.150 -0.010 bb
proteins were placed in an aqueous solution at 10 mg/mL. Proteins Conalbumin Ga -0.100 0.200 0.220 -0.620 -0.080 -1.580 0.160 -0.010 -0.670 0.020 1.040 -0.220 bb
were purified from hen egg whites by carboxymethyl cellulose
Conalbumin Ha 0.540 -0.060 -0.040 -0.020 -0.990 -0.290 0.200 0.160 -0.110 -0.050 -0.560 0.150 bb
chromatography1.
  Ovomucin -0.810 0.020 0.170 0.270 -0.690 0.520 0.040 0.700 0.120 1.700 -0.090 1.060 0 pAb
Spectrophotometry The absorption spectra were measured with a
Different degrees of glycosylation of the same protein.
a Varian Cary 50 Bio UV-Visible Spectrophotometer. Settings were b
Not measured.
dual-beam and medium speed, with a range of 500 to 220 nm. For c
P. DeMoss and A. Fraser.
each protein-lipid combination, a set of three scans was taken and
recorded on a single wavelength vs. absorbance graph: “lipid”, “lipid
= Protein = Protein = Protein
with protein”, and “protein”. For a lipid scan, 566.7 L of lipid = Protein in lipid = Protein in lipid = Protein in lipid
solution was added to a 1 mL cuvette, and then further diluted with
333.3 L of water to a final concentration of .444 mg/mL. The
sample was then scanned. For a lipid with protein scan, 100 L of
protein solution was added to the same solution in the cuvette,
having final concentrations of 1 mg/mL for the protein and .4 mg/mL
of lipid. After scanning, the cuvette was then rinsed thoroughly with
water, and then dried with acetone. For the protein scan, 100 L
protein was added to the same cleaned cuvette, diluted with 900 L wavelength (nm) wavelength (nm) wavelength (nm)
water to a final concentration of 1 mg/mL, and then scanned. A B
  C
Fluorometry The excitation and emission spectra were measured Second derivative absorbance spectra of ovomucoid (A), ovalbumin A 3 (B), and ovoinhibitor (C) in DOPS.
with a Perkin Elmer LS 50 B Luminescence Spectrometer. Settings
for excitation spectra were a slit width of 2.5, and wavelengths from
250-290 nm with an interval of 0.5. Settings for emission spectra
were a slit width of 2.5, and wavelengths from 310-420 nm with an Discussion References
interval of 0.5. The emission and excitation maxima were gained For any particular protein selected, the microenvironment shifts differ [1] Rhodes, M. B. et al., J. Biol. Chem. 230, 399-408 (1958).
from preliminary scans of the proteins. For protein scans, 100 μL of extensively with the type of lipid in ways that correlate neither with lipid head [2] Savitzky, A. and Golay, M. J. E., Anal. Chem. 36, 1627-1639 (1964).
protein solution was added to a 1 mL cuvette, then further diluted group charge nor with acyl chain. By the Kyte-Doolittle hydrophobicity scale, [3] Ichikawa, T. and Terada, H., Biochim. Biophys. Acta 494, 267-270 (1977).
with 900 μL of water, for a final concentration of 1 mg/mL. After the none of these proteins has appreciable hydrophobic character. However, ion [4] Mach, H. and Middaugh, C. R., Anal. Biochem. 222, 323-331 (1994).
sample was scanned and cuvette was washed, 900 μL of lipid channel current measurements show that some of these proteins either [5] Mach, H. et al., Arch. Biochem. Biophys. 287, 33-40 (1991).
solution was scanned. 100 μL of protein was then added, for a final transport ions across a synthetic membrane or cause micropore formation in [6] Lucas, L. H., et al., Protein Sci., 15, 2228-2243 (2006).
lipid concentration of 1.08 mg/mL and a final protein concentration of a synthetic membrane. Spectrophotometric measurements alone have not
1 mg/mL. been sufficient to discern differences between those glycosylated proteins
that do carry monovalent cations across the membrane and those that do
Spectral Processing Second derivatives of spectra were computed not.
by the five-point cubic convolution of Savitzky and Golay 2.
Microenvironments of aromatic residues were estimated from
changes in characteristic absorbance bands3-6.