Crystallographic data collection and refinement statistics.

<p>Values for the highest resolution shell are shown in parentheses.</p

Conformational and dynamical asymmetry of the homodimer.

<p>(A) Superposition of all crystallographically independent CpxA_HDC dimers present in the five different crystal forms reported in this study. The main conformational difference between the distinct CpxA_HDC dimers consists in the position of one CA domain (left in the figure) relative to an invariant region containing both DHp domains and the second CA domain. (B) Phos-tag gel retardation autophosphorylation assay. The assay was performed with 10 µM CpxA_HDC and 1 mM ATP in 20 mM Hepes buffer (pH 7.6), 100 mM NaCl, 50 mM KCl, and 5 mM MgCl₂ at 25°C. At the indicated time points, 15 µl aliquots were removed and mixed with SDS loading buffer. Phospho-proteins were separated by Phos-tag acrylamide gel electrophoresis. (C) Total amount of CpxA_HDC-P and CpxA_HDC in each band was determined by densitometry analysis. The continuous lines were the best fits of the data to a single exponential term.</p

Identification of intragenic suppressors.

<p>A plasmid encoding CpxA_ΔP, a constitutive kinase CpxA variant lacking the sensor domain, was mutagenized and transformed into a <i>cpxA</i> null P_cpxP–<i>lacZ recA</i> strain background. The table shows the CpxA activity conferred by single mutant <i>cpxA</i> alleles in the NS54 strain. Point mutations were identified from colonies displaying a Lac⁻ phenotype in X-gal containing plates.</p

Mechanical model for HK autophosphorylation control.

<p>The inactive kinase conformation (left) involves a putative symmetric conformation of CpxA [modeled from the structures of HK853 (PDB ID 2C2A) and wild-type Af1503 HAMP-EnvZ DHp chimera (PDB ID 3ZWR)], in which the two CA domains are sequestered in a nonproductive DHp–CA complex. The active kinase state (right, as observed in the trigonal crystal form) displays a highly asymmetric conformation of the HK homodimer. Propagated by conformational changes in the HAMP domain, the input signal induces a stress on the central DHp helices, promoting segmental helical motions that result in a strong dynamical asymmetry: one of the CA domains is highly mobile and can form a competent active site, whereas the second CA domain is retained in an inactive conformation by extended hydrophobic interactions with the DHp domain. The insert on the right shows these segmental helical movements (without the CA domains for clarity), in which each color represents a distinct rigid-body rotational movement. As a consequence of these movements, a gap broadens between two helices (indicated by a black arrow) and allows the partial exposure of core hydrophobic residues that contribute to sequester the second CA domain in an inactive conformation.</p

Autophosphorylating Michaelis complex.

<p>(A) Close view of the active site showing the residues directly involved in catalysis, as revealed by the hexagonal crystal structure of the CpxA_{HDC_M228V}–AMPPNP complex (a very similar active site architecture was observed for the trigonal and hexagonal crystal structures of wild-type CpxA_HDC in complex with ATP, obtained at lower resolution). The regions corresponding to the conserved N and H boxes are highlighted in dark green and yellow, respectively. (B) Activation of His248 for phosphoryl transfer. Hydrogen bonding interactions between the imidazole ring of His248, the adjacent acidic residue in the CA domain (Glu249) acting as a general base, and a polar residue from the DHp domain (Asn356) contribute to activate His248 for nucleophilic attack to γ-P of ATP. (C) Schematic representation of the CpxA homodimer illustrating the <i>trans</i>-autophosphorylation reaction. (D) Anchoring of Phe403 and Leu419 to the DHp four-helix bundle represented by its electrostatic surface. The DHp-sequestered (yellow) and mobile (green) CA domains are shown in cartoon representation.</p

HAMP structure and function.

<p>(A) Side and top views of the dimeric HAMP four-helix bundle as observed in the trigonal crystal form. Core hydrophobic residues are shown in stick representation. (B) Crick angle deviation plot of the HAMP helices, as calculated with program samCC <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001776#pbio.1001776-DuninHorkawicz1" target="_blank">[52]</a>. (C) The ability of wild-type and mutant <i>cpxA</i> bacteria to respond to the periplasmic overproduction of either the wild-type MalE (open bars) or the folding-defective mutant MalE31 (light gray bars) and to the presence of 0.2% phenethyl alcohol (dark gray bars) was monitored by measuring β-galactosidase activity from a <i>cpxP</i>–<i>lacZ</i> fusion contained in the NS54 strain expressing the different <i>cpxA</i> alleles. The insert shows the cellular levels of CpxA analyzed by immunobloting membrane protein fractions prepared from the NS54 strain transformed by pLCB (lane 1), pLCBA_wt (lane 2), pLCBA₁₉₇ (lane 3), and pLCBA₂₂₈ (lane 4). The additional band observed in the immunoblot is a cross-reacting protein recognized by the antiserum that serves as a loading control. (D) Phosphotransferase activity of CpxA. Both full-length CpxA and CpxA_M228V proteins (10 µM) were first allowed to autophosphorylate for 20 min at 25°C in the presence of 1 mM ATP, and then an equimolar amount of CpxR_N (N-terminal receiver domain) was added to the reactions. Samples were removed at the indicated time points, and phospho-proteins were separated by Phos-tag acrylamide gel electrophoresis. (E) Autokinase activity of full-length CpxA and CpxA_M228V proteins as determined using radioactive ATP. It is worth noting that only a small fraction of the CpxA–Brij35 complex (∼0.1%) is phosphorylated at steady state, as estimated by PhosTag gels run under the same conditions used in the radioactive assays (unpublished data).</p

Overall structure of CpxA.

<p>(A) Linear representation of the prototypical CpxA domain organization. CpxA is an integral membrane receptor with a periplasmic sensor region (residues 29 to 163) flanked by two transmembrane helices (TM1 and TM2). TM2 connects the sensor domain to the cytosolic transmitter core (residues 188 to 457) formed by three domains: HAMP, DHp, and CA, rainbow colored from N-terminus–C-terminus (blue-red). (B) Cartoon representation of the CpxA_HDC homodimer in the trigonal crystal form. The homodimer is highly asymmetric due to helical kinks nearby Ser238 and Pro253 in helix α₂ (shown in stick representation) and large differences in the positioning and orientation of the two CA domains (shown in surface representation) with respect to the central DHp helical core.</p

Evidence of Unfolded Protein Translocation through a Protein Nanopore

Protein nanopores are mainly used to study transport, unfolding, intrinsically disordered proteins, protein-pore interactions, and protein–ligand complexes. This single-molecule sensor for biomedical and biotechnological applications is promising but until now direct proof of protein translocation through a narrow channel is lacking. Here, we report the translocation of a chimera molecule through the aerolysin nanopore in the presence of a denaturing agent, guanidium chloride (1.5 M) and KCl (1 M). The chimera molecule is composed of the recombinant MalE protein with a unique cysteine residue at the C-terminal position covalently linked to a single-stranded DNA oligonucleotide. Real-time polymerase chain reaction (PCR) was used to detect the presence of chimera molecules that have been effectively translocated from the <i>cis</i> to <i>trans</i> chamber of the set up. Comparing the electrical signature of the chimera related to the protein or oligonucleotide alone demonstrates that each type of molecule displays different dynamics in term of transport time, event frequency, and current blockade. This original approach provides the possibility to study protein translocation through different biological, artificial, and biomimetic nanopores or nanotubes. New future applications are now conceivable such as protein refolding at the nanopore exit, peptides and protein sequencing, and peptide characterization for diagnostics

Wild Type, Mutant Protein Unfolding and Phase Transition Detected by Single-Nanopore Recording

Understanding protein folding remains a challenge. A difficulty is to investigate experimentally all the conformations in the energy landscape. Only single molecule methods, fluorescence and force spectroscopy, allow observing individual molecules along their folding pathway. Here we observe that single-nanopore recording can be used as a new single molecule method to explore the unfolding transition and to examine the conformational space of native or variant proteins. We show that we can distinguish unfolded states from partially folded ones with the aerolysin pore. The unfolding transition curves of the destabilized variant are shifted toward the lower values of the denaturant agent compared to the wild type protein. The dynamics of the partially unfolded wild type protein follows a first-order transition. The denaturation curve obtained with the aerolysin pore is similar to that obtained with the α-hemolysin pore. The nanopore geometry or net charge does not influence the folding transition but changes the dynamics

Thermal Unfolding of Proteins Probed at the Single Molecule Level Using Nanopores

The nanopore technique has great potential to discriminate conformations of proteins. It is a very interesting system to mimic and understand the process of translocation of biomacromolecules through a cellular membrane. In particular, the unfolding and folding of proteins before and after going through the nanopore are not well understood. We study the thermal unfolding of a protein, probed by two protein nanopores: aerolysin and α-hemolysin. At room temperature, the native folded protein does not enter into the pore. When we increase the temperature from 25 to 50 °C, the molecules unfold and the event frequency of current blockade increases. A similar sigmoid function fits the normalized event frequency evolution for both nanopores, thus the unfolding curve does not depend on the structure and the net charge of the nanopore. We performed also a circular dichroism bulk experiment. We obtain the same melting temperature (around 45 °C) using the bulk and single molecule techniques