Membrane proteins are key components of living cells. They constitute one-third of the ORFs of virtually all genomes, perform crucial roles, and are targeted by a huge number of drugs.
When present in their native environment, transmembrane proteins are inserted via hydrophobic segments in a lipidic bilayer. Their in vitro characterization requires the extraction of proteins from the membrane and the maintenance of them in a soluble and native state. This is generally achieved using amphiphilic compounds, termed detergents, yielding so-called protein-detergent complexes (pdc). Several membrane proteins are naturally found as oligomers, and maintaining this precise assembly is a crucial issue for further studies.
In this article, we describe a method to determine the quaternary structure of membrane proteins and to follow its retention during protein handling. It is well known that classical size-exclusion chromatography (SEC) using column calibration does not apply in the case of pdc, whose volume and shape also depend on the detergent fraction. Light scattering coupled to chromatography is a promising technique, it does not suffer from the usual bias affecting batch measurements, and it allows the evaluation of sample homogeneity, oligomerization state, protein-protein interaction, and complex formation with other chemical species.
An illustration of this method is provided by the quaternary structure study of the Methanosarcina mazei CorA transporter in two detergents. Crystallographic studies of a homologous CorA revealed that the protein works as a pentamer to conduct ions across the membrane and constitutes a suitable system to characterize the effect of detergents on its oligomeric state.
We used a combination of UV spectrophotometry (UV), multi-angle light scattering (MALS), and refractometry, coupled on-line with an analytical SEC column to analyze both integrity and quaternary structure of purified CorA in two different detergents (Figure 1).
UV, MALS, and refractometry measurements were achieved with a Photo Diode Array 2996 (Waters), a MiniDAWN TREOS (Wyatt Technology) and an Optilab rEX (Wyatt), respectively. We used a 15 mL KW-804 column (Shodex) run at 0.5 mL/min-1 on an Alliance HPLC 2695 system (Waters). The buffer used was 10 mM Tris (pH 8.0), 300 mM NaCl, 5% (v/v) glycerol and 0.05% n-dodecyl-b-D-maltopyranoside (DDM) or 0.1% lauryl-dimethyl-N-amine-oxide (LDAO).
The Zimm’s formalism for the static light-scattering eqation gives:
where R(Θ,c) is the excess Rayleigh ratio of the solution as a function of scattering angle Θ and the solute weight concentration c. It is directly proportional to the intensity of the scattered light in excess of the light scattered by the pure solvent.
Mw is the weight-averaged solute molar mass. A2 is the second virial coefficient in the virial expansion of the osmotic pressure. P(Θ) describes the angular dependence of the scattered light and can be related to the radius of gyration. K* is the constant 4π2(dn/dc)2n02/Naλ04. Na is Avogadro’s number; dn/dc is the refractive index increment with molecular concentration; n0 is the solvent refractive index and l0 is the wavelength of the incident light.
P(Θ) approximately equals 1 in the case of biopolymers. The weight concentration c of the solute, for soluble nonconjugated proteins, can be measured on-line either with optical density at 280 nm or with refractometry. At the low concentrations usually encountered in SEC, the virial coefficient term is negligible. For protein alone, dn/dc is constant and is comprised of between 0.18 and 0.185 mL/g-1.
In the case of membrane proteins, a two-component analysis can be carried out to quantify the proportions of protein and detergent. We achieved this analysis using ASTRA software (Wyatt).
For a pdc, one cannot directly measure the concentration (cpdc) of the eluted species, and the refractive index increment with molecular concentration (dn/dcpdc) is not known a priori. The two terms correspond to the weighted sum of protein and detergent contributions:
where α is the weight proportion of the protein in the pdc, cprot, and cdet are the respective weight concentrations of protein and detergent, and (dn/dc)prot and (dn/dc)det are refractive index increments with molecular concentration, respectively. We used values of 0.133 and 0.148 mL/g-1 for (dn/dc)det of DDM and LDAO, respectively.
Using equation 2, the concentration of the pdc can be expressed as follows:
where n is the measured refractive index difference between pdc solution and solvent.
Similarly, the Beer-Lambert equation can be developed as the sum of contributions from both protein and detergent in the pdc:
where OD280 is the optical density at 280 nm; εpdc, εprot, and εdet are the extinction coefficients at 280 nm, expressed in g/L-1, for the pdc, the protein alone and the detergent, respectively. εprot was theoretically calculated from the protein sequence and εdet is assumed to be 0.003 (Anatrace data sheet).
cpdc and α are unknown in this “two equations with two unknown parameters” system formed by equations 4 and 5. The combined use of UV and refractometry allows one to solve this system and to get values for cpdc and α for each point of the chromatogram. cpdc is then used in equation 1 and the Mw is thus calculated for each point of the chromatogram thanks to the MALS. Mw values for both protein and detergent fractions are finally deduced using the value of α.