ABSTRACT
Single-cell profiling methods have had a profound impact on the understanding of cellular heterogeneity. While genomes and transcriptomes can be explored at the single-cell level, single-cell profiling of proteomes is not yet established. Here we describe new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell profiling. These technologies will in turn facilitate biological discovery and open new avenues for ultrasensitive disease diagnostics.
Subject(s)
Sequence Analysis, Protein/methods , Single Molecule Imaging/methods , Mass Spectrometry/methods , Nanotechnology , Proteins/chemistry , Proteomics/methods , Sequence Analysis, RNA/methods , Single-Cell Analysis/methodsABSTRACT
In vivo proteins fold mainly as they emerge from the ribosome or as they emerge from a membrane translocon. Membrane translocation in particular poses technical challenges to the study of the associated protein folding processes. Recently we have developed a single-molecule methodology that allows the capture of a single protein molecule through a membrane translocon with biotinylated oligonucleotides covalently bound at its N- and C- terminus using streptavidin. The resulting rotaxane can be driven forwards and backwards changing the voltage polarity, and carefully planned experiments allow inference of the folding pathway. Here we will discuss the details of a simplified methodological approach.
Subject(s)
Protein Folding , Proteins , Oligonucleotides/metabolism , Protein Transport , Ribosomes/metabolism , StreptavidinABSTRACT
A technology capable of sequencing individual protein molecules would revolutionize our understanding of biological processes. Nanopore technology can analyze single heteropolymer molecules such as DNA by measuring the ionic current flowing through a single nanometer hole made in an electrically insulating membrane. This current is sensitive to the monomer sequence. However, proteins are remarkably complex and identifying a single residue change in a protein remains a challenge. In this work, I show that simple neural networks can be trained to recognize protein mutants. Although these networks are quickly and efficiently trained, their ability to generalize in an independent experiment is poor. Using a thermal annealing protocol on the nanopore sample, and examining many mutants with the same nanopore sensor are measures aimed at reducing training data variability which produce an increase in the generalizability of the trained neural network. Using this approach, we obtain a 100% correct assignment among 9 mutants in >50% of the experiments. Interestingly, the neural network performance, compared to a random guess, improves as more mutants are included in the dataset for discrimination. Engineered nanopores prepared with high homogeneity coupled with state-of-the-art analysis of the ionic current signals may enable single-molecule protein sequencing.
Subject(s)
Biosensing Techniques , Nanopores , Amino Acids , Nanotechnology , Neural Networks, ComputerABSTRACT
Nanopore technology enables the detection and analysis of single protein molecules. The technique measures the ionic current passing through a single pore inserted in an electrically insulating membrane. The translocation of the protein molecule through the pore causes a modulation of the ionic current. Analysis of the ionic current reveals the biophysics of co-translocational unfolding and may be used to infer the amino acid sequence and posttranslational modifications of the molecule.
Subject(s)
Electrophysiology/methods , Hemolysin Proteins/chemistry , Hemolysin Proteins/metabolism , Ion Channels , Nanopores , Nanotechnology/methods , Oligonucleotides/metabolism , Oligonucleotides/chemistry , Protein Processing, Post-Translational , Protein Unfolding , Staphylococcus aureus/metabolismABSTRACT
Protein post-translational translocation is found at the plasma membrane of prokaryotes and protein import into organellae. Translocon structures are becoming available, however the dynamics of proteins during membrane translocation remain largely obscure. Here we study, at the single-molecule level, the folding landscape of a model protein while forced to translocate a transmembrane pore. We use a DNA tag to drive the protein into the α-hemolysin pore under a quantifiable force produced by an applied electric potential. Using a voltage-quench approach we find that the protein fluctuates between the native state and an intermediate in the translocation process at estimated forces as low as 1.9 pN. The fluctuation kinetics provide the free energy landscape as a function of force. We show that our stable, ≈15 kBT, substrate can be unfolded and translocated with physiological membrane potentials and that selective divalent cation binding may have a profound effect on the translocation kinetics.
Subject(s)
Bacterial Toxins/metabolism , Cell Membrane/metabolism , Escherichia coli Proteins/metabolism , Hemolysin Proteins/metabolism , Thioredoxins/metabolism , Bacterial Toxins/chemistry , Bacterial Toxins/genetics , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Hemolysin Proteins/chemistry , Hemolysin Proteins/genetics , Kinetics , Membrane Potentials , Mutation , Protein Folding , Protein Transport , Protein Unfolding , Single Molecule Imaging , Structure-Activity Relationship , Thioredoxins/chemistry , Thioredoxins/geneticsABSTRACT
Understanding protein folding under conditions similar to those found in vivo remains challenging. Folding occurs mainly vectorially as a polypeptide emerges from the ribosome or from a membrane translocon. Protein folding during membrane translocation is particularly difficult to study. Here, we describe a single-molecule method to characterize the folded state of individual proteins after membrane translocation, by monitoring the ionic current passing through the pore. We tag both N and C termini of a model protein, thioredoxin, with biotinylated oligonucleotides. Under an electric potential, one of the oligonucleotides is pulled through a α-hemolysin nanopore driving the unfolding and translocation of the protein. We trap the protein in the nanopore as a rotaxane-like complex using streptavidin stoppers. The protein is subjected to cycles of unfolding-translocation-refolding switching the voltage polarity. We find that the refolding pathway after translocation is slower than in bulk solution due to the existence of kinetic traps.
Subject(s)
Bacterial Toxins/metabolism , Cell Membrane/metabolism , Escherichia coli Proteins/metabolism , Hemolysin Proteins/metabolism , Rotaxanes/metabolism , Thioredoxins/metabolism , Bacterial Toxins/chemistry , Bacterial Toxins/genetics , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Hemolysin Proteins/chemistry , Hemolysin Proteins/genetics , Kinetics , Membrane Potentials , Membranes, Artificial , Protein Domains , Protein Folding , Protein Transport , Protein Unfolding , Rotaxanes/chemistry , Single Molecule Imaging , Structure-Activity Relationship , Thioredoxins/chemistry , Thioredoxins/geneticsABSTRACT
Obtaining artificial proteins that mimic the DNA binding properties of natural transcription factors could open new ways of manipulating gene expression at will. In this context it is particularly interesting to develop simple synthetic systems. Inspired by the modularity of natural transcription factors, we have designed synthetic miniproteins that combine the zinc finger module of the transcription factor GAGA and AT-hook peptide domains. These constructs are capable of binding to composite DNA sequences of up to 14 base pairs with high affinity and good selectivity. In particular, we have synthesized three different chimeras and characterized their DNA binding properties by electrophoresis and fluorescence anisotropy. We have also used, for the first time in the study of peptide-based DNA binders, nanopore force spectroscopy to obtain further data on the DNA interaction.
ABSTRACT
Protein interactions with specific DNA sequences are crucial in the control of gene expression and the regulation of replication. Single-molecule methods offer excellent capabilities to unravel the mechanism and kinetics of these interactions. Here, we develop a nanopore approach where a target DNA sequence is contained in a hairpin followed by a ssDNA. This system allows DNA-protein complexes to be distinguished from bare DNA molecules as they are pulled through a single nanopore detector, providing both equilibrium and kinetic information. We show that this approach can be used to test the inhibitory effect of small molecules on complex formation and their mechanisms of action. In a proof of concept, we use DNAs with different sequence patterns to probe the ability of the nanopore to distinguish the effects of an inhibitor in a complex mixture of target DNAs and proteins. We anticipate that the use of this technology with arrays of thousands of nanopores will contribute to the development of transcription factor binding inhibitors.
Subject(s)
Biosensing Techniques/methods , DNA/metabolism , Transcription Factors/metabolism , Animals , DNA/chemistry , DNA, Single-Stranded/chemistry , DNA, Single-Stranded/metabolism , Drug Evaluation, Preclinical/methods , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Models, Molecular , Nanopores/ultrastructure , Nanotechnology/methods , Nucleic Acid Conformation , Protein Binding/drug effects , RabbitsABSTRACT
Protein unfolding and translocation through pores occurs during trafficking between organelles, protein degradation and bacterial toxin delivery. In vivo, co-translocational unfolding can be affected by the end of the polypeptide that is threaded into the pore first. Recently, we have shown that co-translocational unfolding can be followed in a model system at the single-molecule level, thereby unravelling molecular steps and their kinetics. Here, we show that the unfolding kinetics of the model substrate thioredoxin, when pulled through an α-haemolysin pore, differ markedly depending on whether the process is initiated from the C terminus or the N terminus. Further, when thioredoxin is pulled from the N terminus, the unfolding pathway bifurcates: some molecules finish unfolding quickly, while others finish ~100 times slower. Our findings have important implications for the understanding of biological unfolding mechanisms and in the application of nanopore technology for the detection of proteins and their modifications.
Subject(s)
Bacterial Toxins/metabolism , Hemolysin Proteins/metabolism , Nanopores , Protein Unfolding , Thioredoxins/metabolism , Humans , Kinetics , Models, Molecular , Protein Folding , Protein TransportABSTRACT
We demonstrate single-molecule, site-specific detection of protein phosphorylation with protein nanopore technology. A model protein, thioredoxin, was phosphorylated at two adjacent sites. Analysis of the ionic current amplitude and noise, as the protein unfolds and moves through an α-hemolysin pore, enables the distinction between unphosphorylated, monophosphorylated and diphosphorylated variants. Our results provide a step toward nanopore proteomics.
Subject(s)
Biotechnology/methods , Nanopores , Phosphorylation , Proteins/analysis , Proteins/chemistry , Amino Acid Sequence , Models, Molecular , Molecular Sequence Data , Proteins/metabolism , Proteomics , Sequence AlignmentABSTRACT
Porins are ß-barrel outer-membrane proteins through which small solutes and metabolites diffuse that are also exploited during cell death. We have studied how the bacteriocin colicin E9 (ColE9) assembles a cytotoxic translocon at the surface of Escherichia coli that incorporates the trimeric porin OmpF. Formation of the translocon involved ColE9's unstructured N-terminal domain threading in opposite directions through two OmpF subunits, capturing its target TolB on the other side of the membrane in a fixed orientation that triggers colicin import. Thus, an intrinsically disordered protein can tunnel through the narrow pores of an oligomeric porin to deliver an epitope signal to the cell to initiate cell death.
Subject(s)
Colicins/metabolism , Escherichia coli/metabolism , Porins/metabolism , Cell Membrane/metabolism , Colicins/chemistry , Colicins/isolation & purification , Escherichia coli/chemistry , Escherichia coli Proteins/metabolism , Periplasmic Proteins/metabolism , Protein Multimerization , Protein Structure, Tertiary , Protein TransportABSTRACT
Cells are divided into compartments and separated from the environment by lipid bilayer membranes. Essential molecules are transported back and forth across the membranes. We have investigated how folded proteins use narrow transmembrane pores to move between compartments. During this process, the proteins must unfold. To examine co-translocational unfolding of individual molecules, we tagged protein substrates with oligonucleotides to enable potential-driven unidirectional movement through a model protein nanopore, a process that differs fundamentally from extension during force spectroscopy measurements. Our findings support a four-step translocation mechanism for model thioredoxin substrates. First, the DNA tag is captured by the pore. Second, the oligonucleotide is pulled through the pore, causing local unfolding of the C terminus of the thioredoxin adjacent to the pore entrance. Third, the remainder of the protein unfolds spontaneously. Finally, the unfolded polypeptide diffuses through the pore into the recipient compartment. The unfolding pathway elucidated here differs from those revealed by denaturation experiments in solution, for which two-state mechanisms have been proposed.
Subject(s)
Nanopores , Protein Unfolding , Animals , Bacterial Toxins/chemistry , Electricity , Escherichia coli/metabolism , Hemolysin Proteins/chemistry , Kinetics , Lipid Bilayers/chemistry , Models, Molecular , Mutation/genetics , Oligonucleotides/chemistry , Protein Unfolding/drug effects , Rabbits , Thioredoxins/chemistry , Thioredoxins/metabolism , Urea/pharmacologyABSTRACT
Mammalian 14-3-3 protein scaffolds include seven conserved isoforms that bind numerous phosphorylated protein partners and regulate many cellular processes. Some 14-3-3-isoforms, notably γ, have elevated affinity for membranes, which might contribute to modulate the subcellular localization of the partners and substantiate the importance of investigating molecular mechanisms of membrane interaction. By applying surface plasmon resonance we here show that the binding to phospholipid bilayers is stimulated when 14-3-3γ is complexed with its partner, a peptide corresponding to the Ser19-phosphorylated N-terminal region of tyrosine hydroxylase. Moreover, membrane interaction is dependent on salts of kosmotropic ions, which also stabilize 14-3-3γ. Electrostatic analysis of available crystal structures of γ and of the non-membrane-binding ζ-isoform, complemented with molecular dynamics simulations, indicate that the electrostatic potential distribution of phosphopeptide-bound 14-3-3γ is optimal for interaction with the membrane through amphipathic helices at the N-terminal dimerization region. In addition, His158, and especially His195, both specific to 14-3-3γ and located at the convex lateral side, appeared to be pivotal for the ligand induced membrane interaction, as corroborated by site-directed mutagenesis. The participation of these histidine residues might be associated to their increased protonation upon membrane binding. Overall, these results reveal membrane-targeting motifs and give insights on mechanisms that furnish the 14-3-3γ scaffold with the capacity for tuned shuffling from soluble to membrane-bound states.
Subject(s)
14-3-3 Proteins/chemistry , 14-3-3 Proteins/metabolism , Cell Membrane/metabolism , Histidine/chemistry , 14-3-3 Proteins/genetics , Amino Acid Sequence , Humans , Hydrogen-Ion Concentration , Ligands , Molecular Docking Simulation , Molecular Dynamics Simulation , Molecular Sequence Data , Mutagenesis, Site-Directed , Phosphopeptides/chemistry , Phosphopeptides/metabolism , Protein Binding , Protein Conformation , Protein Isoforms , Protein Stability , Static Electricity , Surface Plasmon ResonanceABSTRACT
Protein physicochemical properties must undergo complex changes during evolution, as a response to modifications in the organism environment, the result of the proteins taking up new roles or because of the need to cope with the evolution of molecular interacting partners. Recent work has emphasized the role of stability and stability-function trade-offs in these protein adaptation processes. In the present study, on the other hand, we report that combinations of a few conservative, high-frequency-of-fixation mutations in the thioredoxin molecule lead to largely independent changes in both stability and the diversity of catalytic mechanisms, as revealed by single-molecule atomic force spectroscopy. Furthermore, the changes found are evolutionarily significant, as they combine typically hyperthermophilic stability enhancements with modulations in function that span the ranges defined by the quite different catalytic patterns of thioredoxins from bacterial and eukaryotic origin. These results suggest that evolutionary protein adaptation may use, in some cases at least, the potential of conservative mutations to originate a multiplicity of evolutionarily allowed mutational paths leading to a variety of protein modulation patterns. In addition the results support the feasibility of using evolutionary information to achieve protein multi-feature optimization, an important biotechnological goal.
Subject(s)
Evolution, Molecular , Mutation , Proteins/chemistry , Proteins/genetics , Calorimetry, Differential Scanning , Chemical Phenomena , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Kinetics , Microscopy, Atomic Force , Models, Molecular , Protein Conformation , Protein Stability , Proteins/metabolism , Thioredoxins/chemistry , Thioredoxins/genetics , Thioredoxins/physiologyABSTRACT
We introduce proteolytic scanning calorimetry, a modification of the differential scanning calorimetry approach to the determination of protein stability in which a proteolytic enzyme (thermolysin) is used to mimic a harsh environment. This methodology allows the straightforward calculation of the rate of irreversible denaturation as a function of temperature and concentration of proteolytic enzyme and, as a result, has the potential to probe efficiently the fundamental biophysical features of protein kinetic stability. In the particular case of Escherichia coli thioredoxin (used as an illustrative example in this article), we find that the rate of irreversible denaturation is determined by 1), the global unfolding mechanism at low thermolysin concentrations, indicating that thermodynamic stability may contribute directly to the kinetic stability of thioredoxin under moderately harsh conditions and 2), the rate of unfolding at high thermolysin concentrations, indicating that the free-energy barrier for unfolding may act as a safety mechanism that ensures significant kinetic stability, even in very harsh environments. This thioredoxin picture, however, is by no means expected to be general and different proteins may show different patterns of kinetic stabilization. Proteolytic scanning calorimetry is particularly well-suited to probe this diversity at a fundamental biophysical level.
Subject(s)
Microscopy, Acoustic/methods , Thioredoxins/chemistry , Kinetics , Protein DenaturationABSTRACT
Recent work has shown that proteins can tolerate hydrophobic-to-ionizable-residue mutations. Here, we provide experimental evidence that the essential properties (pK value, protonation state, local dynamics) of buried ionizable groups in proteins can be efficiently modulated through the rational design of the surface charge distribution, thus paving the way for the protein engineering exploitation of charge burial.
Subject(s)
Ions/chemistry , Proteins/chemistry , Escherichia coli/chemistry , Escherichia coli/genetics , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Models, Molecular , Mutation , Protein Conformation , Proteins/genetics , Static Electricity , Thermodynamics , Thioredoxins/chemistry , Thioredoxins/geneticsABSTRACT
Many enzymes possess, besides their native function, additional promiscuous activities. Proteins with several activities (multipurpose catalysts) may have a wide range of biotechnological and biomedical applications. Natural promiscuity, however, appears to be of limited scope in this context, because the latent (promiscuous) function is often related to the evolved one (sharing the active site and even the chemical mechanism) and its enhancement upon suitable mutations usually brings about a decrease in the native activity. Here we explore the use of computational protein design to overcome these limitations. The high-plasticity positions close to the original ("native") active-site are the most promising candidates for mutations that create a second active-site associated to a new function. To avoid compromising protein folding and native activity, we propose a minimal-perturbation approach based on the combinatorial optimization of, both the de novo catalytic activity and the folding free-energy: essentially, we construct the Pareto Set of optimal stability/promiscuous-function solutions. We validate our approach by introducing a promiscuous esterase activity in E. coli thioredoxin on the basis of mutations at positions close to the native-active-site disulfide-bridge. Native oxidoreductase activity is not compromised and it is, in fact, found to be 1.5-fold enhanced, as determined by an insulin-reduction assay. This work provides general guidelines as to how computational design can be used to expand the scope and applications of protein promiscuity. From a more general viewpoint, it illustrates the potential of multi-objective optimization as the computational analogue of multi-feature natural selection.
Subject(s)
Catalytic Domain/physiology , Computational Biology , Protein Engineering , Proteins/metabolism , Esterases/metabolism , Models, Molecular , Protein Binding , Proteins/chemistry , Substrate Specificity , Thermodynamics , Thioredoxins/chemistry , Thioredoxins/genetics , Thioredoxins/metabolismABSTRACT
A large collection of structural snapshots along a full catalytic cycle of Escherichia coli thioredoxin reductase (TrxR) has been generated and characterized using a combination of theoretical methods. Molecular models were built starting from the available X-ray crystallographic structures of dimeric wild-type TrxR in the flavin-oxidizing conformation and a C135S TrxR mutant enzyme in a flavin-reducing conformation "trapped" by a cross-link between Cys138 of TrxR and Cys32 of C35S mutant thioredoxin (Trx). The transition between these two extreme states, which is shown to be reproduced in a normal mode analysis, as well as natural cofactor binding and dissociation, were simulated for the wild-type species using unrestrained and targeted molecular dynamics following docking of oxidized Trx to reduced TrxR. The whole set of simulations provides a comprehensive structural framework for understanding the mechanism of disulfide reduction in atomic detail and identifying the most likely intermediates that facilitate entry of NADPH and exit of NADP(+). The crucial role assigned to Arg73 and Lys36 of Trx in substrate binding and complex stabilization was ascertained when R73G, R73D, and K36A site-directed mutants of Trx were shown to be impaired to different extents in their ability to be reduced by TrxR. On the basis of previous findings and the results reported herein, E. coli TrxR appears as a beautifully engineered molecular machine that is capable of synchronizing cofactor capture and ejection with substrate binding and redox activity through an interdomain twisting motion.
Subject(s)
Escherichia coli/enzymology , Thioredoxin-Disulfide Reductase/metabolism , Thioredoxins/metabolism , Binding Sites , Calorimetry, Differential Scanning , Crystallography, X-Ray , Molecular Dynamics Simulation , NADP/metabolism , Point Mutation , Protein Binding , Protein Conformation , Substrate Specificity , Thioredoxin-Disulfide Reductase/chemistry , Thioredoxin-Disulfide Reductase/genetics , Thioredoxins/chemistry , Thioredoxins/geneticsABSTRACT
Tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of catecholamines, is activated by phosphorylation-dependent binding to 14-3-3 proteins. The N-terminal domain of TH is also involved in interaction with lipid membranes. We investigated the binding of the N-terminal domain to its different partners, both in the unphosphorylated (TH-(1-43)) and Ser(19)-phosphorylated (THp-(1-43)) states by surface plasmon resonance. THp-(1-43) showed high affinity for 14-3-3 proteins (K(d) approximately 0.5 microM for 14-3-3gamma and -zeta and 7 microM for 14-3-3eta). The domains also bind to negatively charged membranes with intermediate affinity (concentration at half-maximal binding S(0.5) = 25-58 microM (TH-(1-43)) and S(0.5) = 135-475 microM (THp-(1-43)), depending on phospholipid composition) and concomitant formation of helical structure. 14-3-3gamma showed a preferential binding to membranes, compared with 14-3-3zeta, both in chromaffin granules and with liposomes at neutral pH. The affinity of 14-3-3gamma for negatively charged membranes (S(0.5) = 1-9 microM) is much higher than the affinity of TH for the same membranes, compatible with the formation of a ternary complex between Ser(19)-phosphorylated TH, 14-3-3gamma, and membranes. Our results shed light on interaction mechanisms that might be relevant for the modulation of the distribution of TH in the cytoplasm and membrane fractions and regulation of L-DOPA and dopamine synthesis.
Subject(s)
14-3-3 Proteins/chemistry , Cell Membrane/metabolism , Tyrosine 3-Monooxygenase/chemistry , Amino Acid Sequence , Chromaffin Cells/cytology , Humans , Hydrogen-Ion Concentration , Kinetics , Levodopa/chemistry , Molecular Sequence Data , Phosphorylation , Protein Binding , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Surface Plasmon ResonanceABSTRACT
Thioredoxins (Trxs) are oxidoreductase enzymes, present in all organisms, that catalyze the reduction of disulfide bonds in proteins. By applying a calibrated force to a substrate disulfide, the chemical mechanisms of Trx catalysis can be examined in detail at the single-molecule level. Here we use single-molecule force-clamp spectroscopy to explore the chemical evolution of Trx catalysis by probing the chemistry of eight different Trx enzymes. All Trxs show a characteristic Michaelis-Menten mechanism that is detected when the disulfide bond is stretched at low forces, but at high forces, two different chemical behaviors distinguish bacterial-origin from eukaryotic-origin Trxs. Eukaryotic-origin Trxs reduce disulfide bonds through a single-electron transfer reaction (SET), whereas bacterial-origin Trxs show both nucleophilic substitution (S(N)2) and SET reactions. A computational analysis of Trx structures identifies the evolution of the binding groove as an important factor controlling the chemistry of Trx catalysis.