Probing membrane protein unfolding with pulse proteolysis

Technical challenges have greatly impeded the investigation of membrane protein folding and unfolding. To develop a new tool that facilitates the study of membrane proteins, we tested pulse proteolysis as a probe for membrane protein unfolding. Pulse proteolysis is a method to monitor protein folding and unfolding, which exploits the significant difference in proteolytic susceptibility between folded and unfolded proteins. This method requires only a small amount of protein and, in many cases, may be used with unpurified proteins in cell lysates. To evaluate the effectiveness of pulse proteolysis as a probe for membrane protein unfolding, we chose Halobacterium halobium bacteriorhodopsin (bR) as a model system. The denaturation of bR in SDS has been investigated extensively by monitoring the change in the absorbance at 560 nm (A₅₆₀). In this work, we demonstrate that denaturation of bR by SDS results in a significant increase in its susceptibility to proteolysis by subtilisin. When pulse proteolysis was applied to bR incubated in varying concentrations of SDS, the remaining intact protein determined by electrophoresis shows a cooperative transition. The midpoint of the cooperative transition (C_m) shows excellent agreement with that determined by A₅₆₀. The C_m values determined by pulse proteolysis for M56A and Y57A bRs are also consistent with the measurements made by A₅₆₀. Our results suggest that pulse proteolysis is a quantitative tool to probe membrane protein unfolding. Combining pulse proteolysis with Western blotting may allow the investigation of membrane protein unfolding in situ without overexpression or purification.     

Revisiting absorbance at 230 nm as a protein unfolding probe

Thermodynamic stability and unfolding kinetics of proteins are typically determined by monitoring protein unfolding with spectroscopic probes, such as circular dichroism (CD) and fluorescence. UV absorbance at 230 nm (A₂₃₀) is also known to be sensitive to protein conformation. However, its feasibility for quantitative analysis of protein energetics has not been assessed. Here we evaluate A₂₃₀ as a structural probe to determine thermodynamic stability and unfolding kinetics of proteins. By using Escherichia coli maltose binding protein (MBP) and E. coli ribonuclease H (RNase H) as our model proteins, we monitored their unfolding in urea and guanidinium chloride with A₂₃₀. Significant changes in A₂₃₀ were observed with both proteins on unfolding in the chemical denaturants. The global stabilities were successfully determined by measuring the change in A₂₃₀ in varying concentrations of denaturants. Also, unfolding kinetics was investigated by monitoring the change in A₂₃₀ under denaturing conditions. The results were quite consistent with those determined by CD. Unlike CD, A₂₃₀ allowed us to monitor protein unfolding in a 96-well microtiter plate with a UV plate reader. Our finding suggests that A₂₃₀ is a valid and convenient structural probe to determine thermodynamic stability and unfolding kinetics of proteins with many potential applications.     

The genetic incorporation of p-azidomethyl-l-phenylalanine into proteins in yeast

The noncanonical amino acid p-azidomethyl-l-phenylalanine can be genetically incorporated into proteins in bacteria, and has been used both as a spectroscopic probe and for the selective modification of proteins by alkynes using click chemistry. Here we report identification of Escherichia coli tyrosyl tRNA synthetase mutants that allow incorporation of p-azidomethyl-l-phenylalanine into proteins in yeast. When expressed together with the cognate E. coli tRNA_CUA^Tyr, the new mutant tyrosyl tRNA synthetases directed robust incorporation of p-azidomethyl-l-phenylalanine into a model protein, human superoxide dismutase, in response to the UAG amber nonsense codon. Mass spectrometry analysis of purified superoxide dismutase proteins confirmed the efficient site-specific incorporation of p-azidomethyl-l-phenylalanine. This work provides an additional tool for the selective modification of proteins in eukaryotic cells.     

Investigating protein unfolding kinetics by pulse proteolysis

Investigation of protein unfolding kinetics of proteins in crude samples may provide many exciting opportunities to study protein energetics under unconventional conditions. As an effort to develop a method with this capability, we employed “pulse proteolysis” to investigate protein unfolding kinetics. Pulse proteolysis has been shown to be an effective and facile method to determine global stability of proteins by exploiting the difference in proteolytic susceptibilities between folded and unfolded proteins. Electrophoretic separation after proteolysis allows monitoring protein unfolding without protein purification. We employed pulse proteolysis to determine unfolding kinetics of E. coli maltose binding protein (MBP) and E. coli ribonuclease H (RNase H). The unfolding kinetic constants determined by pulse proteolysis are in good agreement with those determined by circular dichroism. We then determined an unfolding kinetic constant of overexpressed MBP in a cell lysate. An accurate unfolding kinetic constant was successfully determined with the unpurified MBP. Also, we investigated the effect of ligand binding on unfolding kinetics of MBP using pulse proteolysis. On the basis of a kinetic model for unfolding of MBP•maltose complex, we have determined the dissociation equilibrium constant (K_d) of the complex from unfolding kinetic constants, which is also in good agreement with known K_d values of the complex. These results clearly demonstrate the feasibility and the accuracy of pulse proteolysis as a quantitative probe to investigate protein unfolding kinetics.     

Sequence complexity of the S receptor kinase gene family in Brassica

A genomic library from an S ₂₉/S ₂₉ self-incompatible genotype of Brassica oleracea was screened with a probe carrying part of the catalytic domain of a Brassica S-receptor kinase (SRK)-like gene. Six positive phage clones with varying hybridisation intensities (K1 to K6) were purified and characterised. A 650–700 by region corresponding to the probe was excised from each clone and sequenced. DNA and predicted protein sequence comparisons based on a multiple alignment identified K5 as a pseudogene, whereas the others could encode functional proteins. K3 was found to have lost an intron from its genomic sequence. The six genes display different degrees of sequence similarity and form two distinct clusters in a dendrogram. The 98% similarity between K4 and K6, which extends across intron sequences, suggests that these might be very recently diverged alleles or daughters of a duplication. In addition, K2 showed a comparably high similarity to the probe. Clones K1, K3 and K5 cross-hybridised with an SLG ₂₉ cDNA probe, indicating the presence of upstream receptor domains homologous to the Brassica SLG gene. This suggests that the previously reported S sequence complexity may be ascribed to a large receptor kinase gene family.     

An effective thiol-reactive probe for differential scanning fluorimetry with a standard real-time polymerase chain reaction device

Differential scanning fluorimetry (DSF) is used to assess protein stability, transition states, or the K_d values of various ligands, drug molecules, and antibodies. All fluorescent probes published to date either are incompatible with hydrophobic proteins/ligands, precluding analyses of transmembrane or membrane-associated proteins, or have excitation and detection wavelengths outside the range of real-time polymerase chain reaction (RT–PCR) machines, necessitating the use of dedicated devices. Here, we describe a thiol-reactive probe, BODIPY FL l-cystine (BFC), to overcome both of these shortcomings. The probe supports an inexpensive application of DSF measurements suitable for detection with standard RT–PCR machines in a hydrophilic or hydrophobic environment.     

Ancient Origins of RGK Protein Function: Modulation of Voltage-Gated Calcium Channels Preceded the Protostome and Deuterostome Split

RGK proteins, Gem, Rad, Rem1, and Rem2, are members of the Ras superfamily of small GTP-binding proteins that interact with Ca²⁺ channel β subunits to modify voltage-gated Ca²⁺ channel function. In addition, RGK proteins affect several cellular processes such as cytoskeletal rearrangement, neuronal dendritic complexity, and synapse formation. To probe the phylogenetic origins of RGK protein–Ca²⁺ channel interactions, we identified potential RGK-like protein homologs in genomes for genetically diverse organisms from both the deuterostome and protostome animal superphyla. RGK-like protein homologs cloned from Danio rerio (zebrafish) and Drosophila melanogaster (fruit flies) expressed in mammalian sympathetic neurons decreased Ca²⁺ current density as reported for expression of mammalian RGK proteins. Sequence alignments from evolutionarily diverse organisms spanning the protostome/deuterostome divide revealed conservation of residues within the RGK G-domain involved in RGK protein – Ca_vβ subunit interaction. In addition, the C-terminal eleven residues were highly conserved and constituted a signature sequence unique to RGK proteins but of unknown function. Taken together, these data suggest that RGK proteins, and the ability to modify Ca²⁺ channel function, arose from an ancestor predating the protostomes split from deuterostomes approximately 550 million years ago.     

High-affinity bisubstrate probe for fluorescence anisotropy binding/displacement assays with protein kinases PKA and ROCK

The bisubstrate fluorescent probe ARC-583 (Adc-Ahx-(d-Arg)₆-d-Lys(5-TAMRA)-NH₂) and its application for the characterization of both ATP- and protein/peptide substrate-competitive inhibitors of protein kinases PKA (cyclic AMP-dependent protein kinase) and ROCK (rho kinase) in fluorescence polarization-based assay are described. High affinity of the probe (K_D = 0.48 nM toward PKA) enables its application for the characterization of inhibitors with nanomolar and micromolar potency and determination of the active concentration of the kinase in individual experiments as well as in the high-throughput screening format. The probe can be used for the assessment of protein-protein interactions (e.g., between regulatory and catalytic subunits of PKA) and as a cyclic AMP biosensor.     

Exploring the Denatured State Ensemble by Single-Molecule Chemo-Mechanical Unfolding: The Effect of Force,Temperature, and Urea

While it is widely appreciated that the denatured state of a protein is a heterogeneous conformational ensemble, there is still debate over how this ensemble changes with environmental conditions. Here, we use single-molecule chemo-mechanical unfolding, which combines force and urea using the optical tweezers, together with traditional protein unfolding studies to explore how perturbants commonly used to unfold proteins (urea, force, and temperature) affect the denatured-state ensemble. We compare the urea m-values, which report on the change in solvent accessible surface area for unfolding, to probe the denatured state as a function of force, temperature, and urea. We find that while the urea- and force-induced denatured states expose similar amounts of surface area, the denatured state at high temperature and low urea concentration is more compact. To disentangle these two effects, we use destabilizing mutations that shift the T_m and C_m. We find that the compaction of the denatured state is related to changing temperature as the different variants of acyl-coenzyme A binding protein have similar m-values when they are at the same temperature but different urea concentration. These results have important implications for protein folding and stability under different environmental conditions.     

Studies on beef heart ubiquinol-cytochrome c reductase. Quantification and distribution of the core proteins as determined by radioimmunoassay

Core proteins I (M_r 50 000) and II (M_r 47 000) were isolated from beef heart ubiquinol-cytochrome c reductase, and radioimmunoassays were developed for both. Immunoreplica experiments show that antisera against each protein react with a single peptide in both isolated Complex III and in mitochondria. Thus, core proteins are not aggregated forms of smaller peptides as suggested for the yeast protein (Jeffrey, A., Power, S. and Palmer, G., Biochem. Biophys. Res. Commun. (1979) 86, 271–277). Core proteins were quantitated in Complex III and in mitochondria using radioimmunoassay. Approx. 2 mol core protein II per mol core protein I were found. A molar ratio of 1 : 2 : 2 : 1 is suggested for core protein I : core protein II : cytochrome b : cytochrome c₁. Radioimmunoassay shows that the antibodies react as extensively with Complex III-bound core protein as with the isolated core proteins. In spite of this, the antibodies do not inhibit electron transport in submitochondrial particles or isolated Complex III, and they have no oligomycin- or uncoupler-like effects on submitochondrial particles oxidizing NADH. The combined results from radioimmunoassay and immunoreplica experiments strongly suggest, however, that core proteins are specifically associated with Complex III in the mitochondria, implying a specific role there.     

Coordination of the cell-specific distribution of the four subunits of glycine decarboxylase and of serine hydroxymethyltransferase in leaves of C3-C4 intermediate species from different genera

The cell-specific distribution of the four subunit proteins (P, L, T and H) of glycine decarboxylase (GDC) and of serine hydroxymethyltransferase (SHMT) has been studied in the leaves of C₃-C₄ intermediate and C₄ species of three genera (Flaveria, Moricandia and Panicum) using immunogold localization. Antibodies raised against these proteins from pea leaf mitochondria were used to probe Western blots of total leaf proteins of F. linearis Lag., M. arvensis (L.) DC and P. milioides Nees ex Trin. (C₃-C₄), and F. trinervia (Spring.) Mohr and P. miliaceum (L.) (C₄). For all species, each antibody recognised specifically a protein of similar molecular weight to that in pea leaves. In leaves of M. arvensis the P protein was present in the mitochondria of the bundle-sheath cells but was undetectable in those of the mesophyll, whereas the L, T and H proteins and SHMT were present in both cell types. The density of immunogold labelling of SHMT on the mitochondria of mesophyll cells was less than that on those of the bundle-sheath cells, which correlates with the relative activities of SHMT in these cell types. These data reveal that the lack of functional GDC in the mesophyll cells of M. arvensis, which is the principal biochemical reason for reduced photorespiration in this species, is due to the loss of a single subunit protein. This lack of coordinate expression of the subunit proteins of GDC within a photosynthetic cell represents a clear difference between M. arvensis and other C₃ and C₃-C₄ species. None of the GDC proteins was detectable in the mesophyll cells of the C₃-C₄ and C₄ Flaveria and Panicum species but all were present in the bundle-sheath cells. The differences in the distribution of the GDC proteins in leaves of the C₃-C₄ species studied are discussed in relation to the evolution of photosynthetic mechanisms.     

Comparison of in vitro and in vivo oligomeric states of a wild type and mutant trimeric inner membrane multidrug transporter

Many membrane proteins exist and function as oligomers or protein complexes. Routine analytical methods involve extraction and solubilization of the proteins with detergents, which could disturb their actual oligomeric state. AcrB is a trimeric inner membrane multidrug transporter in E. coli. In previous studies, we created a mutant AcrB_P223G, which behaves like a monomer when extracted from the cell membrane. However, the actual oligomeric state of AcrB_P223G in cell membranes remained unclear, which complicated the interpretation of the mechanism by which the mutation affects function. Here we used several complementary methods to determine the oligomeric state of AcrB_P223G in E. coli cell membranes. Two sets of quantitative fluorescent techniques were exploited. For these, we created fluorescent tagged AcrB, AcrB-CFP and AcrB-YPet. Fluorescence resonance energy transfer (FRET) and fluorescence recovery after photobleaching (FRAP) were employed to characterize independently the efficiency of energy transfer between co-expressed AcrB-CFP and AcrB-YPet, and the diffusion coefficient of AcrB-YPet and AcrB_P223G-YPet in live E. coli cells. Second, we introduced Cys pairs at the inter-subunit interface and used controlled oxidation to probe inter-subunit distances. The results from all studies converge on the conclusion that AcrB_P223G exists as a trimer in cell membranes, which dissociates during the purification steps. The small change in trimer affinity and structure leads to a significant loss of AcrB activity. In addition, throughout this study we developed protocols and established benchmark values, useful for further studies on membrane protein associations in cell membranes.     

Purification of allergenic proteins from peanut for preparation of the reactive solid phase of a specific IgE radioimmunoassay

Peanut is one of the most allergenic foods. Detection of specific IgE in the serum of allergic patients requires the purification of allergenic proteins. In the present work, proteins were recovered from peanut kernel after successive treatment in acetone and diethy ether. The proteins were dissolved in 0.05% TFA and analysed by RP-HPLC with a 0–100% gradient of methanol containing 0.05% TFA. The protein peaks were recovered and tested in SDS-PAGE. Eleven proteins were identified with a M_r ranging from 13 to 81. Western blotting was performed with sera from allergic patients. Allergenic proteins had a M_r of 15, 18, 19, 33, 41 and 67. By comparison, a protein fraction from peanut shell contained seven proteins with M_r ranging from 15 to 81. Only two proteins with M_r of 18 and 41 were detected in a Western blot. The protein fractions were coupled to epoxy-Sepharose and the gels were used as a solid reactive phase for detection by IgE-RIA of specific IgE from the serum of allergic patients.     

A Novel High Throughput Biochemical Assay to Evaluate the HuR Protein-RNA Complex Formation

The RNA binding protein HuR/ELAVL1 binds to AU-rich elements (AREs) promoting the stabilization and translation of a number of mRNAs into the cytoplasm, dictating their fate. We applied the AlphaScreen technology using purified human HuR protein, expressed in a mammalian cell-based system, to characterize in vitro its binding performance towards a ssRNA probe whose sequence corresponds to the are present in TNFα 3’ untranslated region. We optimized the method to titrate ligands and analyzed the kinetic in saturation binding and time course experiments, including competition assays. The method revealed to be a successful tool for determination of HuR binding kinetic parameters in the nanomolar range, with calculated Kd of 2.5±0.60 nM, k _on of 2.76±0.56*10⁶ M^-1 min^-1, and k _off of 0.007±0.005 min^-1. We also tested the HuR-RNA complex formation by fluorescent probe-based RNA-EMSA. Moreover, in a 384-well plate format we obtained a Z-factor of 0.84 and an averaged coefficient of variation between controls of 8%, indicating that this biochemical assay fulfills criteria of robustness for a targeted screening approach. After a screening with 2000 small molecules and secondary verification with RNA-EMSA we identified mitoxantrone as an interfering compound with rHuR and TNFα probe complex formation. Notably, this tool has a large versatility and could be applied to other RNA Binding Proteins recognizing different RNA, DNA, or protein species. In addition, it opens new perspectives in the identification of small-molecule modulators of RNA binding proteins activity.     

ARF-B(2): A Protein Complex that Specifically Binds to Part of the Anaerobic Response Element of Maize Adh 1

Crude whole cell extracts from maize (Zea mays L.) suspension cells were examined for DNA binding proteins that specifically interact with a portion of the maize Adh 1 promoter that was previously shown to be in contact with a trans-acting factor in vivo. A 17 base pair, double-stranded oligonucleotide probe was constructed that centered around a strong in vivo dimethylsulfate footprint (B₂) that coincides with part of the anaerobic response element (ARE). Gel retardation assays were used to characterize a major, specific DNA binding protein activity found in the crude extracts. The activity is present in both aerobic and hypoxically treated cultures and has been designated ARF-B₂ (ARE binding factor). ARF-B₂ appears to be a multicomponent complex, with a 54 kilodalton subunit termed ARF-B₂α in primary contact with the target DNA.     

A genetically encoded sensor for H2O2 with expanded dynamic range

Hydrogen peroxide is an important second messenger controlling intracellular signaling cascades by selective oxidation of redox active thiolates in proteins. Changes in intracellular [H₂O₂] can be tracked in real time using HyPer, a ratiometric genetically encoded fluorescent probe. Although HyPer is sensitive and selective for H₂O₂ due to the properties of its sensing domain derived from the Escherichia coli OxyR protein, many applications may benefit from an improvement of the indicator’s dynamic range. We here report HyPer-2, a probe that fills this demand. Upon saturating [H₂O₂] exposure, HyPer-2 undergoes an up to sixfold increase of the ratio F500/F420 versus a threefold change in HyPer. HyPer-2 was generated by a single point mutation A406V from HyPer corresponding to A233V in wtOxyR. This mutation was previously shown to destabilize interface between monomers in OxyR dimers. However, in HyPer-2, the A233V mutation stabilizes the dimer and expands the dynamic range of the probe.     

Use of immobilized Streptomyces griseus proteases (pronase) as a probe of structural transitions of lysozyme, β-lactoglobulin and casein

The native - denatured (N U) structural transition in lysozyme (mucopeptide N-acetylmuramoylhydrolase, EC 3.2.1.17), β-lactoglobulin and caseins have been studied by proteolysis using immobilized Streptomyces griseus proteases (pronase) as a probe. A diverse range of susceptibility to urea denaturation was revealed by evaluation of initial rates and pseudo first-order rate constants for hydrolysis of these proteins. Comparison of the rate of hydrolysis of lysozyme vis-à-vis performic acid oxidized-lysozyme showed that the degree of backbone accessibility for native lysozyme, even in concentrated urea solutions, was less than that of the oxidized protein. At pH 7.5, native lysozyme appeared to possess the most stable structure, followed by β-lactoglobulin and, finally, the caseins. It is postulated that the proteolytic rate depends upon accessibility of a susceptible bond(s) or subtle conformational changes in the least stable domain. Following cleavage of this bond(s), K_D increases thus exposing more backbone. Use of pronase immobilized on porous succinamidopropyl-glass beads resulted in increased enzyme stability and eliminated autolysis. Consequently, immobilized proteases are an excellent probe of structural transitions of protein substrates in denaturants.     

Frizzled receptors signal through G proteins

Frizzled receptors have long been thought to couple to G proteins but biochemical evidence supporting such an interaction has been lacking. Here we expressed mammalian Wnt-Frizzled fusion proteins in Saccharomyces cerevisiae and tested the receptors' ability to activate the yeast mitogen-activated protein kinase (MAPK) pathway via heterotrimeric G proteins. Our results show that Frizzled receptors can interact with Gα_i, Gα_q, and Gα_s proteins, thus confirming that Frizzled functions as a G protein coupled receptor (GPCR). However, the activity level of Frizzled-mediated G protein signaling was much lower than that of a typical GPCR and, surprisingly, was highest when coupled to Gα_s. The Frizzled/Gα_s interaction was further established in vivo as Drosophila expressing a loss-of-function Gα_s allele rescued the photoreceptor differentiation phenotype of Frizzled mutant flies. Together, these data point to an important role for Frizzled as a nontraditional GPCR that preferentially couples to Gα_s heterotrimeric G proteins.