25 research outputs found
Computational modeling of biological nanopores
Throughout our history, we, humans, have sought to better control and
understand our environment. To this end, we have extended our natural senses
with a host of sensors-tools that enable us to detect both the very large, such
as the merging of two black holes at a distance of 1.3 billion light-years from
Earth, and the very small, such as the identification of individual viral
particles from a complex mixture. This dissertation is devoted to studying the
physical mechanisms that govern a tiny, yet highly versatile sensor: the
biological nanopore. Biological nanopores are protein molecules that form
nanometer-sized apertures in lipid membranes. When an individual molecule
passes through this aperture (i.e., "translocates"), the temporary disturbance
of the ionic current caused by its passage reveals valuable information on its
identity and properties. Despite this seemingly straightforward sensing
principle, the complexity of the interactions between the nanopore and the
translocating molecule implies that it is often very challenging to
unambiguously link the changes in the ionic current with the precise physical
phenomena that cause them. It is here that the computational methods employed
in this dissertation have the potential to shine, as they are capable of
modeling nearly all aspects of the sensing process with near atomistic
precision.
Beyond familiarizing the reader with the concepts and state-of-the-art of the
nanopore field, the primary goals of this dissertation are fourfold: (1)
Develop methodologies for accurate modeling of biological nanopores; (2)
Investigate the equilibrium electrostatics of biological nanopores; (3)
Elucidate the trapping behavior of a protein inside a biological nanopore; and
(4) Mapping the transport properties of a biological nanopore.
In the first results chapter of this thesis (Chapter 3), we used 3D
equilibrium simulations [...]Comment: PhD thesis, 306 pages. Source code available at
https://github.com/willemsk/phdthesis-tex
Engineering and Modeling the Electrophoretic Trapping of a Single Protein Inside a Nanopore
The ability to confine and to study single molecules has enabled important advances in natural and applied sciences. Recently, we have shown that unlabeled proteins can be confined inside the biological nanopore Cytolysin A (ClyA) and conformational changes monitored by ionic current recordings. However, trapping small proteins remains a challenge. Here we describe a system where steric, electrostatic, electrophoretic, and electroosmotic forces are exploited to immobilize a small protein, dihydrofolate reductase (DHFR), inside ClyA. Assisted by electrostatic simulations, we show that the dwell time of DHFR inside ClyA can be increased by orders of magnitude (from milliseconds to seconds) by manipulation of the DHFR charge distribution. Further, we describe a physical model that includes a double energy barrier and the main electrophoretic components for trapping DHFR inside the nanopore. Simultaneous fits to the voltage dependence of the dwell times allowed retrieving direct estimates of the cis and trans translocation probabilities, the mean dwell time, and the force exerted by the electroosmotic flow on the protein (≅9 pN at -50 mV). The observed binding of NADPH to the trapped DHFR molecules suggested that the engineered proteins remained folded and functional inside ClyA. Contact-free confinement of single proteins inside nanopores can be employed for the manipulation and localized delivery of individual proteins and will have further applications in single-molecule analyte sensing and enzymology studies
Electro-osmotic capture and ionic discrimination of peptide and protein biomarkers with FraC nanopores
Biological nanopores are nanoscale sensors employed for high-throughput, low-cost, and long read-length DNA sequencing applications. The analysis and sequencing of proteins, however, is complicated by their folded structure and non-uniform charge. Here we show that an electro-osmotic flow through Fragaceatoxin C (FraC) nanopores can be engineered to allow the entry of polypeptides at a fixed potential regardless of the charge composition of the polypeptide. We further use the nanopore currents to discriminate peptide and protein biomarkers from 25 kDa down to 1.3 kDa including polypeptides differing by one amino acid. On the road to nanopore proteomics, our findings represent a rationale for amino-acid analysis of folded and unfolded polypeptides with nanopores.Biological nanopore-based protein sequencing and recognition is challenging due to the folded structure or non-uniform charge of peptides. Here the authors show that engineered FraC nanopores can overcome these problems and recognize biomarkers in the form of oligopeptides, polypeptides and folded proteins
PlyAB Nanopores Detect Single Amino Acid Differences in Folded Haemoglobin from Blood
The real-time identification of protein biomarkers is crucial for the development of point-of-care and portable devices. Here, we use a PlyAB biological nanopore to detect haemoglobin (Hb) variants. Adult HbA and sickle cell anaemia HbS, which differ by just one amino acid, were distinguished in a mixture with more than 97 % accuracy based on individual blockades. Foetal Hb, which shows a larger sequence variation, was distinguished with near 100 % accuracy. Continuum and Brownian dynamics simulations revealed that Hb occupies two energy minima, one near the inner constriction and one at the trans entry of the nanopore. Thermal fluctuations, the charge of the protein, and the external bias influence the dynamics of Hb within the nanopore, which in turn generates the unique ionic current signal in the Hb variants. Finally, Hb was counted from blood samples, demonstrating that direct discrimination and quantification of Hb from blood using nanopores, is feasible
Accurate modeling of a biological nanopore with an extended continuum framework
Despite the broad success of biological nanopores as powerful instruments for the analysis of proteins and nucleic acids at the single-molecule level, a fast simulation methodology to accurately model their nanofluidic properties is currently unavailable. This limits the rational engineering of nanopore traits and makes the unambiguous interpretation of experimental results challenging. Here, we present a continuum approach that can faithfully reproduce the experimentally measured ionic conductance of the biological nanopore Cytolysin A (ClyA) over a wide range of ionic strengths and bias potentials. Our model consists of the extended Poisson-Nernst-Planck and Navier-Stokes (ePNP-NS) equations and a computationally efficient 2D-axisymmetric representation for the geometry and charge distribution of the nanopore. Importantly, the ePNP-NS equations achieve this accuracy by self-consistently considering the finite size of the ions and the influence of both the ionic strength and the nanoscopic scale of the pore on the local properties of the electrolyte. These comprise the mobility and diffusivity of the ions, and the density, viscosity and relative permittivity of the solvent. Crucially, by applying our methodology to ClyA, a biological nanopore used for single-molecule enzymology studies, we could directly quantify several nanofluidic characteristics difficult to determine experimentally. These include the ion selectivity, the ion concentration distributions, the electrostatic potential landscape, the magnitude of the electro-osmotic flow field, and the internal pressure distribution. Hence, this work provides a means to obtain fundamental new insights into the nanofluidic properties of biological nanopores and paves the way towards their rational engineering
Analysis tools for single-monomer measurements of self-assembly processes
Protein assembly plays an important role throughout all phyla of life, both
physiologically and pathologically. In particular, aggregation and
polymerization of proteins are key-strategies that regulate cellular function.
In recent years, methods to experimentally study the assembly process on a
single-molecule level have been developed. This progress concomitantly has
triggered the question of how to analyze this type of single-filament data
adequately and what experimental conditions are necessary to allow a meaningful
interpretation of the analysis. Here, we developed two analysis methods for
single-filament data: the visitation analysis and the average-rate analysis. We
benchmarked and compared both approaches with the classic dwell-time-analysis
frequently used to study microscopic association and dissociation rates. In
particular, we tested the limitations of each analysis method along the lines
of the signal-to-noise ratio, the sampling rate, and the labeling efficiency
and bleaching rate of the fluorescent dyes used in single-molecule fluorescence
experiments. Finally, we applied our newly developed methods to study the
monomer assembly of actin at the single-molecule-level in the presence of the
class II nucleator Cappuccino and the WH2 repeats of Spire. For Cappuccino, our
data indicated fast elongation circumventing a nucleation phase whereas, for
spire, we found that the four WH2 motifs are not sufficient to promote de novo
nucleation of actin
DNA Translocation through Nanopores at Physiological Ionic Strengths Requires Precise Nanoscale Engineering
Many important processes in biology involve the translocation of a biopolymer through a nanometer-scale pore. Moreover, the electrophoretic transport of DNA across nanopores is under intense investigation for single-molecule DNA sequencing and analysis. Here, we show that the precise patterning of the ClyA biological nanopore with positive charges is crucial to observe the electrophoretic translocation of DNA at physiological ionic strength. Surprisingly, the strongly electronegative 3.3 nm internal constriction of the nanopore did not require modifications. Further, DNA translocation could only be observed from the wide entry of the nanopore. Our results suggest that the engineered positive charges are important to align the DNA in order to overcome the entropic and electrostatic barriers for DNA translocation through the narrow constriction. Finally, the dependencies of nucleic acid translocations on the Debye length of the solution are consistent with a physical model where the capture of double-stranded DNA is diffusion-limited while the capture of single-stranded DNA is reaction-limited.status: publishe
DNA Translocation through Nanopores at Physiological Ionic Strengths Requires Precise Nanoscale Engineering
Single-molecule nanopore enzymology
Biological nanopores are a class of membrane proteins that open nanoscale water conduits in biological membranes. When they are reconstituted in artificial membranes and a bias voltage is applied across the membrane, the ionic current passing through individual nanopores can be used to monitor chemical reactions, to recognize individual molecules and, of most interest, to sequence DNA. In addition, a more recent nanopore application is the analysis of single proteins and enzymes. Monitoring enzymatic reactions with nanopores, i.e. nanopore enzymology, has the unique advantage that it allows long-timescale observations of native proteins at the single-molecule level. Here, we describe the approaches and challenges in nanopore enzymology. This article is part of the themed issue 'Membrane pores: from structure and assembly, to medicine and technology'
DNA Translocation through Nanopores at Physiological Ionic Strengths Requires Precise Nanoscale Engineering
Many important processes in biology involve the translocation of a biopolymer through a nanometer-scale pore. Moreover, the electrophoretic transport of DNA across nanopores is under intense investigation for single-molecule DNA sequencing and analysis. Here, we show that the precise patterning of the ClyA biological nanopore with positive charges is crucial to observe the electrophoretic translocation of DNA at physiological ionic strength. Surprisingly, the strongly electronegative 3.3 nm internal constriction of the nanopore did not require modifications. Further, DNA translocation could only be observed from the wide entry of the nanopore. Our results suggest that the engineered positive charges are important to align the DNA in order to overcome the entropic and electrostatic barriers for DNA translocation through the narrow constriction. Finally, the dependencies of nucleic acid translocations on the Debye length of the solution are consistent with a physical model where the capture of double-stranded DNA is diffusion-limited while the capture of single-stranded DNA is reaction-limited