Acrylamide Concentration Determines the Direction and Magnitude of Helical Membrane Protein Gel Shifts
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Acrylamide concentration determines the direction and magnitude of helical membrane protein gel shifts Arianna Ratha, Fiona Cunninghama,b,1, and Charles M. Debera,b,2 aDivision of Molecular Structure and Function, Research Institute, Hospital for Sick Children, Toronto, ON, Canada M5G 1X8; and bDepartment of Biochemistry, University of Toronto, Toronto, ON, Canada M5S 1A8 Edited by David A. Weitz, Harvard University, Cambridge, MA, and approved August 16, 2013 (received for review June 13, 2013) SDS/PAGE is universally used in biochemistry, cell biology, and (5)]. Larger particles become trapped within the gel meshwork immunology to resolve minute protein amounts readily from and migrate slower than smaller species. Low-percentage gels tissue and cell extracts. Although molecular weights of water- are therefore typically used to resolve larger proteins, and vice soluble proteins are reliably determined from their SDS/PAGE versa. Acrylamide concentrations compatible with routine use mobility, most helical membrane proteins, which comprise 20–30% are usually from 4–20% T due to practical considerations, of the human genome and the majority of drug targets, migrate to because gels outside of this range are too fragile or too brittle, positions that have for decades been unpredictably slower or respectively, to withstand the physical manipulation(s) required faster than their actual formula weight, often confounding their for protein visualization and/or immunoblotting. identification. Using de novo designed transmembrane-mimetic Most globular, water-soluble proteins are reliably identified by polypeptides that match the composition of helical membrane- their SDS/PAGE mobility relative to corresponding reference spanning sequences, we quantitate anomalous SDS/PAGE frac- proteins typically used to estimate molecular weight. However, tionation of helical membrane proteins by comparing the relative this group of well-behaved polypeptides does not include helical mobilities of these polypeptides with typical water-soluble refer- transmembrane (TM) proteins, macromolecules that comprise fi ence proteins on Laemmli gels. We nd that both the net charge 20–30% of the human genome (6), comprise the majority of drug and effective molecular size of the migrating particles of trans- targets (7), and are the focus of major pharmaceutical discovery membrane-mimetic species exceed those of the corresponding ref- efforts (8). For example, the first true G protein-coupled re- erence proteins and that gel acrylamide concentration dictates the ceptor to be determined to high resolution, 39-kDa bovine impact of these two factors on the direction and magnitude of rhodopsin (9), migrates on SDS/PAGE to positions consistent anomalous migration. Algorithms we derived from these data with sizes as low as 30 kDa (10). In fact, we have previously compensate for this differential effect of acrylamide concentration shown that the gel mobility of helical TM proteins seldom cor- on the SDS/PAGE mobility of a variety of natural membrane pro- responds to formula molecular weight (11). This phenomenon of teins. Our results provide a unique means to predict anomalous “anomalous migration” can arise as a consequence of the high migration of membrane proteins, thereby facilitating straightfor- hydrophobicity and concomitant binding of DS by TM proteins ward determination of their molecular weights via SDS/PAGE. at levels that exceed those of water-soluble polypeptides (12). fi However, quantitation of DS binding stoichiometry is not rou- gel mobility | protein migration | protein identi cation | apparent size | fi immunoblotting tine and consumes milligram amounts of puri ed samples. Thus, the impact of enhanced DS binding on the direction and mag- ’ nitude of anomalous migration has remained unpredictable for aemmli s system for polyacrylamide gel protein electropho- decades, with helical TM proteins variously exhibiting gel mo- Lresis in the presence of the detergent SDS (SDS/PAGE) is bility reduced, equivalent, or increased relative to reference one of the most cited methodological papers in life sciences (1). proteins (11–13). Such differences are generally disregarded when The facility with which SDS/PAGE resolves minute amounts of proteins revolutionized the analysis of tissue and cell extracts, Significance resulting in “overnight” adoption of the technique in biochemistry, cell biology, immunology, and virology (2). Considered “the single most useful analytical tool to study protein molecules” (3), SDS/ SDS/PAGE is a protein analysis technique universally used in PAGE is routinely used for simultaneous determination of protein biochemistry, cell biology, immunology, and virology, where heterogeneity and molecular weight in applications ranging from proteins are separated by size on a gel matrix of poly- acrylamide. However, most helical membrane proteins, which diagnosis of hereditary red cell membrane disorders to evaluation – of recombinant protein expression and purification procedures. are biomolecules that comprise 20 30% of genomes and the Protein analysis by SDS/PAGE is relatively simple, affordable, and majority of drug targets, migrate to positions on SDS/PAGE that have for decades been unpredictably larger or smaller rapid (4): A buffer containing a tracking dye and SDS is added to than their actual size. We have found that the magnitude and the sample of interest, the mixture is applied to a polyacrylamide direction of migration among membrane protein mimetics are gel, and a potential difference is used to drive the dye and the controlled by the acrylamide concentration in the gel. Our resulting anionic particle composed of protein and dodecyl sulfate results facilitate straightforward SDS/PAGE analysis of these (DS) through the gel. The distance traveled by the protein/DS important biomolecules. particle from the top of the gel is then divided by that of the dye to obtain relative migration (Rf), and molecular weight [as Author contributions: A.R. and C.M.D. designed research; A.R. and F.C. performed re- relative molecular mass (Mr)] determined by comparison of this search; A.R. contributed new reagents/analytic tools; A.R. and C.M.D. analyzed data; and A.R. and C.M.D. wrote the paper. value with a logarithmic plot derived from the RfsandMrsof reference proteins. The authors declare no conflict of interest. Fractionation on SDS/PAGE is controlled by the molecular This article is a PNAS Direct Submission. size and shape of the protein/DS particle, its net charge, and the 1Present address: Mitacs, York University, Toronto, ON, Canada M3J 1P3. accessible spaces among the acrylamide fibers that comprise 2To whom correspondence should be addressed. E-mail: [email protected]. the gel matrix as determined by the total concentration of acryl- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. amide and bis-acrylamide cross-linker [T; Materials and Methods 1073/pnas.1311305110/-/DCSupplemental. 15668–15673 | PNAS | September 24, 2013 | vol. 110 | no. 39 www.pnas.org/cgi/doi/10.1073/pnas.1311305110 Downloaded by guest on September 28, 2021 protein identity is known or can be confirmed in orthogonal the Tris-glycine system) were selected for the present work molecular weight determination procedures but, in many in- because they are used in the majority of determinations of pro- stances, raise questions of protein folding, oligomeric organiza- tein complexity and molecular weight (16), and are used most tion, proteolytic processing, posttranslational modification(s), often among reported systems in our database of helical TM alternative splicing, antibody cross-reaction, and/or degradation. protein SDS/PAGE mobilities (11) (SI Materials and Methods). These issues become acute in SDS/PAGE analyses of tissue or Gels using these buffers at >10% T resolve reference proteins in cell extracts, where reasonable molecular weight estimates re- the range of ≥14–200 kDa, whereas those with ≥14% T frac- main crucial for protein identification. tionate species ≥3.5 kDa (Mark12 Unstained Standard on Life Here, we quantitate anomalous SDS/PAGE fractionation of Technologies Novex Tris-Glycine Gel SDS/PAGE Migration helical membrane proteins by comparing the relative mobilities Charts, www.invitrogen.com). Given that our TM-mimetics en- of de novo designed TM-mimetic peptide polymers with typical compass the range from 3.5–41 kDa (13), we accordingly chose water-soluble reference proteins on Laemmli gels ranging from to analyze SDS/PAGE migration behavior on gels of 11–18% T, 11–18% T. We find that net charge and effective molecular size in 1% T intervals. among the migrating TM-mimetic species exceed those of the corresponding reference proteins and that gel acrylamide con- Gel Mobility of TM-Mimetics Relative to Reference Proteins Changes centration dictates the impact of these two factors on the direction with Acrylamide Concentration. The set of TM protein mimetics and magnitude of anomalous migration. Algorithms derived from we designed and prepared are polymers of a peptide with the “ ” these data compensate for the differential effect of acrylamide core sequence NH2-SKSKS-Leu20-SKSKS-NH2, termed pL20 concentration on the SDS/PAGE mobility of a variety of natural (13). The average length, high hydrophobicity, and abundance of membrane proteins. Our results provide a straightforward means Leu in natural membrane-spanning regions are recapitulated in to predict anomalous migration of membrane proteins relative to the 20-Leu segment of pL20,