15 research outputs found
Quasithermodynamic Contributions to the Fluctuations of a Protein Nanopore
Proteins undergo thermally activated
conformational fluctuations
among two or more substates, but a quantitative inquiry on their kinetics
is persistently challenged by numerous factors, including the complexity
and dynamics of various interactions, along with the inability to
detect functional substates within a resolvable time scale. Here,
we analyzed in detail the current fluctuations of a monomeric β-barrel
protein nanopore of known high-resolution X-ray crystal structure.
We demonstrated that targeted perturbations of the protein nanopore
system, in the form of loop-deletion mutagenesis, accompanying alterations
of electrostatic interactions between long extracellular loops, produced
modest changes of the differential activation free energies calculated
at 25 °C, ΔΔ<i>G</i><sup><i>⧧</i></sup>, in the range near the thermal energy but substantial and
correlated modifications of the differential activation enthalpies,
ΔΔ<i>H</i><sup>⧧</sup>, and entropies,
ΔΔ<i>S</i><sup>⧧</sup>. This finding
indicates that the local conformational reorganizations of the packing
and flexibility of the fluctuating loops lining the central constriction
of this protein nanopore were supplemented by changes in the single-channel
kinetics. These changes were reflected in the enthalpy–entropy
reconversions of the interactions between the loop partners with a
compensating temperature, <i>T</i><sub>C</sub>, of ∼300
K, and an activation free energy constant of ∼41 kJ/mol. We
also determined that temperature has a much greater effect on the
energetics of the equilibrium gating fluctuations of a protein nanopore
than other environmental parameters, such as the ionic strength of
the aqueous phase as well as the applied transmembrane potential,
likely due to ample changes in the solvation activation enthalpies.
There is no fundamental limitation for applying this approach to other
complex, multistate membrane protein systems. Therefore, this methodology
has major implications in the area of membrane protein design and
dynamics, primarily by revealing a better quantitative assessment
on the equilibrium transitions among multiple well-defined and functionally
distinct substates of protein channels and pores
Sampling a Biomarker of the Human Immunodeficiency Virus across a Synthetic Nanopore
One primary goal in nanobiotechnology is designing new methodologies for molecular biomedical diagnosis at stages much earlier than currently possible and without use of expensive reagents and sophisticated equipment. In this work, we show the proof of principle for single-molecule detection of the nucleocapsid protein 7 (NCp7), a protein biomarker of the HIV-1 virus, using synthetic nanopores and the resistive-pulse technique. The biosensing mechanism relied upon specific interactions between NCp7 and aptamers of stem-loop 3 (SL3) in the packaging domain of the retroviral RNA genome. One critical step of this study was the choice of the optimal size of the nanopores for accurate, label-free determinations of the dissociation constant of the NCp7 protein–SL3 RNA aptamer complex. Therefore, we systematically investigated the NCp7 protein–SL3 RNA aptamer complex employing two categories of nanopores in a silicon nitride membrane: (i) small, whose internal diameter was smaller than 6 nm, and (ii) large, whose internal diameter was in the range of 7 to 15 nm. Here, we demonstrate that only the use of nanopores with an internal diameter that is smaller than or comparable with the largest cross-sectional size of the NCp7–SL3 aptamer complex enables accurate measurement of the dissociation constant between the two interacting partners. Notably, this determination can be accomplished without the need for prior nanopore functionalization. Moreover, using small solid-state nanopores, we demonstrate the ability to detect drug candidates that inhibit the binding interactions between NCp7 and SL3 RNA by using a test case of <i>N</i>-ethylmaleimide
Detergent Desorption of Membrane Proteins Exhibits Two Kinetic Phases
Gradual dissociation of detergent molecules from water-insoluble
membrane proteins culminates in protein aggregation. However, the
time-dependent trajectory of this process remains ambiguous because
the signal-to-noise ratio of most spectroscopic and calorimetric techniques
is drastically declined by the presence of protein aggregates in solution.
We show that by using steady-state fluorescence polarization (FP)
spectroscopy the dissociation of the protein–detergent complex
(PDC) can be inspected in real time at detergent concentrations below
the critical micelle concentration. This article provides experimental
evidence of the coexistence of two distinct phases of the dissociations
of detergent monomers from membrane proteins. We first noted a slow
detergent predesolvation process, which was accompanied by a relatively
modest change in the FP anisotropy, suggesting a small number of dissociated
detergent monomers from the proteomicelles. This predesolvation phase
was followed by a fast detergent desolvation process, which was highlighted
by a major alteration in the FP anisotropy. The durations and rates
of these phases were dependent on both the detergent concentration
and the interfacial PDC interactions. Further development of this
approach might lead to the creation of a new semiquantitative method
for the assessment of the kinetics of association and dissociation
of proteomicelles
Aberrantly Large Single-Channel Conductance of Polyhistidine Arm-Containing Protein Nanopores
There have been only
a few studies reporting on the impact of polyhistidine
affinity tags on the structure, function, and dynamics of proteins.
Because of the relatively short size of the tags, they are often thought
to have little or no effect on the conformation or activity of a protein.
Here, using membrane protein design and single-molecule electrophysiology,
we determined that the presence of a hexahistidine arm at the N-terminus
of a truncated FhuA-based protein nanopore, leaving the C-terminus
untagged, produces an unusual increase in the unitary conductance
to ∼8 nS in 1 M KCl. To the best of our knowledge, this is
the largest single-channel conductance ever recorded with a monomeric
β-barrel outer membrane protein. The hexahistidine arm was captured
by an anti-polyhistidine tag monoclonal antibody added to the side
of the channel-forming protein addition, but not to the opposite side,
documenting that this truncated FhuA-based protein nanopore inserts
into a planar lipid bilayer with a preferred orientation. This finding
is in agreement with the protein insertion <i>in vivo</i>, in which the large loops face the extracellular side of the membrane.
The aberrantly large single-channel conductance, likely induced by
a greater cross-sectional area of the pore lumen, along with the vectorial
insertion into a lipid membrane, will have profound implications for
further developments of engineered protein nanopores
An Outer Membrane Protein Undergoes Enthalpy- and Entropy-Driven Transitions
β-Barrel membrane proteins often fluctuate among
various
open substates, yet the nature of these transitions is not fully understood.
Using temperature-dependent, single-molecule electrophysiology analysis,
along with rational protein design, we show that OccK1, a member of
the outer membrane carboxylate channel from <i>Pseudomonas aeruginosa</i>, features a discrete gating dynamics comprising both enthalpy-driven
and entropy-driven current transitions. OccK1 was chosen for the analysis
of these transitions, because it is a monomeric transmembrane β-barrel
of a known high-resolution crystal structure and displays three distinguishable,
time-resolvable open substates. Native and loop-deletion OccK1 proteins
showed substantial changes in the activation enthalpies and entropies
of the channel transitions, but modest alterations in the equilibrium
free energies, confirming that the system never departs from equilibrium.
Moreover, some current fluctuations of OccK1 indicated a counterintuitive,
negative activation enthalpy, which was compensated by a significant
decrease in the activation entropy. Temperature scanning of the single-channel
properties of OccK1 exhibited a thermally induced switch of the energetically
most favorable open substate at the lowest examined temperature of
4 °C. Therefore, such a semiquantitative assessment of the current
fluctuation dynamics not only demonstrates the complexity of channel
gating but also reveals distinct functional traits of a β-barrel
outer membrane protein under different temperature circumstances
OccK Channels from <i>Pseudomonas aeruginosa</i> Exhibit Diverse Single-Channel Electrical Signatures but Conserved Anion Selectivity
<i>Pseudomonas aeruginosa</i> is a Gram-negative
bacterium
that utilizes substrate-specific outer membrane (OM) proteins for
the uptake of small, water-soluble nutrients employed in the growth
and function of the cell. In this paper, we present for the first
time a comprehensive single-channel examination of seven members of
the OM carboxylate channel K (OccK) subfamily. Recent biochemical,
functional, and structural characterization of the OccK proteins revealed
their common features, such as a closely related, monomeric, 18-stranded β-barrel
conformation with a kidney-shaped transmembrane pore and the presence
of a basic ladder within the channel lumen. Here, we report that the
OccK proteins exhibited fairly distinct unitary conductance values,
in a much broader range than previously expected, which includes low
(∼40–100 pS) and medium (∼100–380 pS)
conductance. These proteins showed diverse single-channel dynamics
of current gating transitions, revealing one-open substate (OccK3),
two-open substate (OccK4–OccK6), and three-open substate (OccK1,
OccK2, and OccK7) kinetics with functionally distinct conformations.
Interestingly, we discovered that anion selectivity is a conserved
trait among the members of the OccK subfamily, confirming the presence
of a net pool of positively charged residues within their central
constriction. Moreover, these results are in accord with an increased
specificity and selectivity of these protein channels for negatively
charged, carboxylate-containing substrates. Our findings might ignite
future functional examinations and full atomistic computational studies
for unraveling a mechanistic understanding of the passage of small
molecules across the lumen of substrate-specific, β-barrel OM
proteins
Representative single-channel electrical recordings of the members of the OccD and OccK subfamilies with solved crystal structures.
<p>(A) OccD1; (B) OccD2; (C) OccD3; (D) OccK1; (E) OccK2; (F) OccK3; (G) OccK4; (H) OccK5; and (I) OccK6. The data were collected at an applied transmembrane potential of +80 mV. The buffer solution in the chamber contained 1 M KCl, 10 mM potassium phosphate, pH = 7.4. For the sake of clarity, the single-channel electrical traces were low-pass Bessel filtered at 2 kHz. The numbers located above the traces represent the single-channel conductance of the most probable substate of the channel. The averages were derived from at least three independent single-channel electrical recordings.</p
Definition of Occ channel specificity.
<p>Uptake of radiolabeled arginine by OccD1, benzoate by OccK1, and glucuronate by OccK2 was measured in the presence of a 10-fold excess of unlabeled low-molecular weight compounds. In all cases, total levels of uptake are reported, expressed as a percentage of uptake in the absence of unlabeled compound (100%). Substrates containing a carboxyl group are italicized.</p>a<p>Reported values are the average of two or three experiments.</p>b<p>Efficient inhibition values, defined as resulting in <50% transport, are shown in bold.</p
Small substrate-specific pores can mediate efficient substrate uptake.
<p>Comparison of pore sizes of OccD1 (PDB ID: 2 ODJ) and OccK1 with those of the non-specific porins OmpG and OmpF. The surface views are shown from the extracellular side.</p
Binding and transport of antibiotics by Occ channels is very specific.
<p>Effect on arginine (A–C), benzoate (E), glucuronate (F), and pyroglutamate (G) uptake in the presence of a 10-fold excess of various antibiotics. (A) OccD1, (B) OccD2, (C) OccD3, (E) OccK1, (F) OccK2, and (G) OccK3. In addition, representative liposome swelling assays are shown with imipenem (D) and cefotaxime (H).</p