Arianna Rath

Detergent binding explains anomalous SDS-PAGE migration of membrane proteins

Migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) that does not correlate with formula molecular weights, termed “gel shifting,” appears to be common for membrane proteins but has yet to be conclusively explained. In the present work, we investigate the anomalous gel mobility of helical membrane proteins using a library of wild-type and mutant helix-loop-helix (“hairpin”) sequences derived from transmembrane segments 3 and 4 of the human cystic fibrosis transmembrane conductance regulator (CFTR), including disease-phenotypic residue substitutions. We find that these hairpins migrate at rates of −10% to +30% vs. their actual formula weights on SDS-PAGE and load detergent at ratios ranging from 3.4–10 g SDS/g protein. We additionally demonstrate that mutant gel shifts strongly correlate with changes in hairpin SDS loading capacity (R2 = 0.8), and with hairpin helicity (R2 = 0.9), indicating that gel shift behavior originates in altered detergent binding. In some cases, this differential solvation by SDS may result from replacing protein-detergent contacts with protein-protein contacts, implying that detergent binding and folding are intimately linked. The CF-phenotypic V232D mutant included in our library may thus disrupt CFTR function via altered protein-lipid interactions. The observed interdependence between hairpin migration, SDS aggregation number, and conformation additionally suggests that detergent binding may provide a rapid and economical screen for identifying membrane proteins with robust tertiary and/or quaternary structures.

The structure of “unstructured” regions in peptides and proteins: Role of the polyproline II helix in protein folding and recognition*

Classical descriptions of the three‐dimensional shapes of proteins usually invoke three main structures: α‐helix, β‐sheet, and β‐turn. More recently, the polyproline II (PPII) structure has been implicated in diverse biological activities including signal transduction, transcription, cell motility, and immune response. Concurrently, evidence is accumulating that PPII structure has a significant role in the unfolded states of proteins. In this article, we connect the structural properties of PPII helices to their roles in protein recognition and protein unfolded states. The properties unique to the PPII conformation are linked to the exploitation of this structure for the molecular recognition of proteins, using peptide ligands of the Src homology 3 (SH3) domain as an example. The evidence supporting a role for PPII conformation in protein‐unfolded states is also presented in the context of the forces that may stabilize the PPII conformation in unfolded polypeptides.

Peptide Models of Membrane Protein Folding

Given the central roles of membrane proteins in cellular processes ranging from nutrient uptake to cell-cell communication, as well as the importance of these proteins as drug targets, efforts to understand and control their structures are vital in human health and disease. The rational design of membrane proteins with modified properties is thus a highly desirable goal in molecular medicine and biotechnology. However, experimental data showing how individual transmembrane (TM) residues and/or segments direct the packing and folding of membrane proteins into biologically functional entities remain sparse. To address these questions in a systematic manner, helix-helix interactions between two (or more) TM segments must be identified and analyzed. Here we present an overview of the utilization of peptides as models of the TM segments of alpha-helical membrane proteins in uncovering the amino acid sequence motifs and interactions that build these molecules. TM peptide design and production strategies are discussed, and specific examples of the application of TM peptides to the study of membrane proteins are presented. We demonstrate that TM peptides can be routinely produced in sufficient quantities for biophysical analysis, are amenable to a variety of experimental techniques, and can effectively replicate the native helix-helix contacts and key aspects of the natural biological structures of membrane proteins.

Evidence for assembly of small multidrug resistance proteins by a "two-faced" transmembrane helix.

Clinically significant bacterial resistance to drugs and cytotoxic compounds can be conferred by the energy-dependent efflux of toxicants, catalyzed by proteins embedded in the bacterial cell membrane. One such group of proteins, the small multidrug resistance family, are drug/proton antiporters that must oligomerize to function, a process that requires the assembly of at least two inactive monomers by intermolecular association of their four transmembrane helices. Here, we have used peptides that correspond to each of the four wild type transmembrane helices of the Halobacterium salinarum protein Hsmr and a corresponding library of mutant peptides to determine the interactive surfaces that likely contribute to protein oligomerization. Hsmr peptides were examined for strong (sodium dodecyl sulfate-resistant) and weaker (perfluorooctanoate-resistant) helix-helix interactions, in conjunction with circular dichroism, fluorescence energy transfer measurements, and molecular modeling. The results are compatible with a scheme in which two faces of helix four permit self-assembly via a higher affinity asymmetric pairing and a lower affinity symmetric interaction, resulting in a discrete tetramer. Our finding that two surfaces of helix four can contribute to the stability of small multidrug resistance protein assembly provides a molecular basis for the design of therapeutics that target this antibiotic resistance mechanism.

Acrylamide concentration determines the direction and magnitude of helical membrane protein gel shifts

Significance SDS/PAGE is a protein analysis technique universally used in biochemistry, cell biology, immunology, and virology, where proteins are separated by size on a gel matrix of polyacrylamide. However, most helical membrane proteins, which are biomolecules that comprise 20–30% of genomes and the majority of drug targets, migrate to positions on SDS/PAGE that have for decades been unpredictably larger or smaller than their actual size. We have found that the magnitude and direction of migration among membrane protein mimetics are controlled by the acrylamide concentration in the gel. Our results facilitate straightforward SDS/PAGE analysis of these important biomolecules. SDS/PAGE is universally used in biochemistry, cell biology, and immunology to resolve minute protein amounts readily from tissue and cell extracts. Although molecular weights of water-soluble proteins are reliably determined from their SDS/PAGE mobility, most helical membrane proteins, which comprise 20–30% of the human genome and the majority of drug targets, migrate to positions that have for decades been unpredictably slower or faster than their actual formula weight, often confounding their identification. Using de novo designed transmembrane-mimetic polypeptides that match the composition of helical membrane-spanning sequences, we quantitate anomalous SDS/PAGE fractionation of helical membrane proteins by comparing the relative mobilities of these polypeptides with typical water-soluble reference proteins on Laemmli gels. We find that both the net charge and effective molecular size of the migrating particles of transmembrane-mimetic species exceed those of the corresponding reference proteins and that gel acrylamide concentration dictates the impact of these two factors on the direction and magnitude of anomalous migration. Algorithms we derived from these data compensate for this differential effect of acrylamide concentration on the SDS/PAGE mobility of a variety of natural membrane proteins. Our results provide a unique means to predict anomalous migration of membrane proteins, thereby facilitating straightforward determination of their molecular weights via SDS/PAGE.

Protein Structure in Membrane Domains

Of great interest to the academic and pharmaceutical research communities, helical transmembrane proteins are characterized by their ability to dissolve and fold in lipid bilayers--properties conferred by polypeptide spans termed transmembrane domains (TMDs). The apolar nature of TMDs necessitates the use of membrane-mimetic solvents for many structure and folding studies. This review examines the relationship between TMD structure and solvent environment, focusing on principles elucidated largely in membrane-mimetic environments with single-TMD protein and peptide models. Following a brief description of TMD sequence and conformational characteristics gleaned from the structural database, we present an overview of the conceptual models used to study folding in vitro. The impact of sequence and solvent context on the incorporation of TMDs into membranes, and its role in measurements of TMD self-assembly strengths, is then described. We conclude with a discussion of the nonspecific effects of membrane components on TMD stability.

The Biologically Relevant Targets and Binding Affinity Requirements for the Function of the Yeast Actin-Binding Protein 1 Src-Homology 3 Domain Vary With Genetic Context

Many protein–protein interaction domains bind to multiple targets. However, little is known about how the interactions of a single domain with many proteins are controlled and modulated under varying cellular conditions. In this study, we investigated the in vivo effects of Abp1p SH3 domain mutants that incrementally reduce target-binding affinity in four different yeast mutant backgrounds in which Abp1p activity is essential for growth. Although the severity of the phenotypic defects observed generally increased as binding affinity was reduced, some genetic backgrounds (prk1Δ and sla1Δ) tolerated large affinity reductions while others (sac6Δ and sla2Δ) were much more sensitive to these reductions. To elucidate the mechanisms behind these observations, we determined that Ark1p is the most important Abp1p SH3 domain interactor in prk1Δ cells, but that interactions with multiple targets, including Ark1p and Scp1p, are required in the sac6Δ background. We establish that the Abp1p SH3 domain makes different, functionally important interactions under different genetic conditions, and these changes in function are reflected by changes in the binding affinity requirement of the domain. These data provide the first evidence of biological relevance for any Abp1p SH3 domain-mediated interaction. We also find that considerable reductions in binding affinity are tolerated by the cell with little effect on growth rate, even when the actin cytoskeletal morphology is significantly perturbed.

Structural, Functional, and Bioinformatic Studies Demonstrate the Crucial Role of an Extended Peptide Binding Site for the SH3 Domain of Yeast Abp1p

SH3 domains, which are among the most frequently occurring protein interaction modules in nature, bind to peptide targets ranging in length from 7 to more than 25 residues. Although the bulk of studies on the peptide binding properties of SH3 domains have focused on interactions with relatively short peptides (less than 10 residues), a number of domains have been recently shown to require much longer sequences for optimal binding affinity. To gain greater insight into the binding mechanism and biological importance of interactions between an SH3 domain and extended peptide sequences, we have investigated interactions of the yeast Abp1p SH3 domain (AbpSH3) with several physiologically relevant 17-residue target peptide sequences. To obtain a molecular model for AbpSH3 interactions, we solved the structure of the AbpSH3 bound to a target peptide from the yeast actin patch kinase, Ark1p. Peptide target complexes from binding partners Scp1p and Sjl2p were also characterized, revealing that the AbpSH3 uses a common extended interface for interaction with these peptides, despite Kd values for these peptides ranging from 0.3 to 6 μm. Mutagenesis studies demonstrated that residues across the whole 17-residue binding site are important both for maximal in vitro binding affinity and for in vivo function. Sequence conservation analysis revealed that both the AbpSH3 and its extended target sequences are highly conserved across diverse fungal species as well as higher eukaryotes. Our data imply that the AbpSH3 must bind extended target sites to function efficiently inside the cell.

The Assembly Motif of a Bacterial Small Multidrug Resistance Protein

Multidrug transporters such as the small multidrug resistance (SMR) family of bacterial integral membrane proteins are capable of conferring clinically significant resistance to a variety of common therapeutics. As antiporter proteins of ∼100 amino acids, SMRs must self-assemble into homo-oligomeric structures for efflux of drug molecules. Oligomerization centered at transmembrane helix four (TM4) has been implicated in SMR assembly, but the full complement of residues required to mediate its self-interaction remains to be characterized. Here, we use Hsmr, the 110-residue SMR family member of the archaebacterium Halobacterium salinarum, to determine the TM4 residue motif required to mediate drug resistance and SMR self-association. Twelve single point mutants that scan the central portion of the TM4 helix (residues 85–104) were constructed and were tested for their ability to confer resistance to the cytotoxic compound ethidium bromide. Six residues were found to be individually essential for drug resistance activity (Gly90, Leu91, Leu93, Ile94, Gly97, and Val98), defining a minimum activity motif of 90GLXLIXXGV98 within TM4. When the propensity of these mutants to dimerize on SDS-PAGE was examined, replacements of all but Ile resulted in ∼2-fold reduction of dimerization versus the wild-type antiporter. Our work defines a minimum activity motif of 90GLXLIXXGV98 within TM4 and suggests that this sequence mediates TM4-based SMR dimerization along a single helix surface, stabilized by a small residue heptad repeat sequence. These TM4-TM4 interactions likely constitute the highest affinity locus for disruption of SMR function by directly targeting its self-assembly mechanism.

Congenital Heart Block Maternal Sera Autoantibodies Target an Extracellular Epitope on the α1G T-Type Calcium Channel in Human Fetal Hearts

Background Congenital heart block (CHB) is a transplacentally acquired autoimmune disease associated with anti-Ro/SSA and anti-La/SSB maternal autoantibodies and is characterized primarily by atrioventricular (AV) block of the fetal heart. This study aims to investigate whether the T-type calcium channel subunit α1G may be a fetal target of maternal sera autoantibodies in CHB. Methodology/Principal Findings We demonstrate differential mRNA expression of the T-type calcium channel CACNA1G (α1G gene) in the AV junction of human fetal hearts compared to the apex (18–22.6 weeks gestation). Using human fetal hearts (20–22 wks gestation), our immunoprecipitation (IP), Western blot analysis and immunofluorescence (IF) staining results, taken together, demonstrate accessibility of the α1G epitope on the surfaces of cardiomyocytes as well as reactivity of maternal serum from CHB affected pregnancies to the α1G protein. By ELISA we demonstrated maternal sera reactivity to α1G was significantly higher in CHB maternal sera compared to controls, and reactivity was epitope mapped to a peptide designated as p305 (corresponding to aa305–319 of the extracellular loop linking transmembrane segments S5–S6 in α1G repeat I). Maternal sera from CHB affected pregnancies also reacted more weakly to the homologous region (7/15 amino acids conserved) of the α1H channel. Electrophysiology experiments with single-cell patch-clamp also demonstrated effects of CHB maternal sera on T-type current in mouse sinoatrial node (SAN) cells. Conclusions/Significance Taken together, these results indicate that CHB maternal sera antibodies readily target an extracellular epitope of α1G T-type calcium channels in human fetal cardiomyocytes. CHB maternal sera also show reactivity for α1H suggesting that autoantibodies can target multiple fetal targets.