Proteins are the active workhorses in our body. These biomolecules perform all vital cellular functions from DNA replication and general biosynthesis to metabolic signaling and environmental sensing.While static 3D structures are now readily available, observing the functional cycle of proteins-involving conformational changes and interactions-remains very challenging, e.g., due to ensemble averaging. However, time-resolved information is crucial to gain a mechanistic understanding of protein function. Single-molecule techniques such as FRET and force spectroscopies provide answers but can be limited by the required labelling, a narrow time bandwidth, and more. Here, we describe electrical nanopore detection as a tool for probing protein dynamics. With a time bandwidth ranging from microseconds to hours, nanopore experiments cover an exceptionally wide range of timescales that is very relevant for protein function. First, we discuss the working principle of label-free nanopore experiments, various pore designs, instrumentation, and the characteristics of nanopore signals. In the second part, we review a few nanopore experiments that solved research questions in protein science, and we compare nanopores to other single-molecule techniques. We hope to make electrical nanopore sensing more accessible to the biochemical community, and to inspire new creative solutions to resolve a variety of protein dynamics-one molecule at a time.

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This paper provides a perspective on potential applications of a new single-molecule technique, viz., the nanopore electro-osmotic trap (NEOtrap). This solid-state nanopore-based method uses locally induced electro-osmosis to form a hydrodynamic trap for single molecules. Ionic current recordings allow one to study an unlabeled protein or nanoparticle of arbitrary charge that can be held in the nanopore's most sensitive region for very long times. After motivating the need for improved single-molecule technologies, we sketch various possible technical extensions and combinations of the NEOtrap. We lay out diverse applications in biosensing, enzymology, protein folding, protein dynamics, fingerprinting of proteins, detecting post-translational modifications, and all that at the level of single proteins – illustrating the unique versatility and potential of the NEOtrap.

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Many strategies have been pursued to trap and monitor single proteins over time to detect the molecular mechanisms of these essential nanomachines. Single-protein sensing with nanopores is particularly attractive because it allows label-free high-bandwidth detection on the basis of ion currents. Here we present the nanopore electro-osmotic trap (NEOtrap) that allows trapping and observing single proteins for hours with submillisecond time resolution. The NEOtrap is formed by docking a DNA-origami sphere onto a passivated solid-state nanopore, which seals off a nanocavity of a user-defined size and creates an electro-osmotic flow that traps nearby particles irrespective of their charge. We demonstrate the NEOtrap’s ability to sensitively distinguish proteins on the basis of size and shape, and discriminate between nucleotide-dependent protein conformations, as exemplified by the chaperone protein Hsp90. Given the experimental simplicity and capacity for label-free single-protein detection over the broad bio-relevant time range, the NEOtrap opens new avenues to study the molecular kinetics underlying protein function.

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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.

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Nanopores bear great potential as single-molecule tools for bioanalytical sensing and sequencing, due to their exceptional sensing capabilities, high-throughput, and low cost. The detection principle relies on detecting small differences in the ionic current as biomolecules traverse the nanopore. A major bottleneck for the further progress of this technology is the noise that is present in the ionic current recordings, because it limits the signal-to-noise ratio (SNR) and thereby the effective time resolution of the experiment. Here, we review the main types of noise at low and high frequencies and discuss the underlying physics. Moreover, we compare biological and solid-state nanopores in terms of the SNR, the important figure of merit, by measuring translocations of a short ssDNA through a selected set of nanopores under typical experimental conditions. We find that SiN_x solid-state nanopores provide the highest SNR, due to the large currents at which they can be operated and the relatively low noise at high frequencies. However, the real game-changer for many applications is a controlled slowdown of the translocation speed, which for MspA was shown to increase the SNR > 160-fold. Finally, we discuss practical approaches for lowering the noise for optimal experimental performance and further development of the nanopore technology.

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Single-molecule Förster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For many molecular machines in a cell proteins have to act together with interaction partners in a functional cycle to fulfill their task. The extension of two-color to multi-color smFRET makes it possible to simultaneously probe more than one interaction or conformational change. This not only adds a new dimension to smFRET experiments but it also offers the unique possibility to directly study the sequence of events and to detect correlated interactions when using an immobilized sample and a total internal reflection fluorescence microscope (TIRFM). Therefore, multi-color smFRET is a versatile tool for studying biomolecular complexes in a quantitative manner and in a previously unachievable detail. Here, we demonstrate how to overcome the special challenges of multi-color smFRET experiments on proteins. We present detailed protocols for obtaining the data and for extracting kinetic information. This includes trace selection criteria, state separation, and the recovery of state trajectories from the noisy data using a 3D ensemble Hidden Markov Model (HMM). Compared to other methods, the kinetic information is not recovered from dwell time histograms but directly from the HMM. The maximum likelihood framework allows us to critically evaluate the kinetic model and to provide meaningful uncertainties for the rates. By applying our method to the heat shock protein 90 (Hsp90), we are able to disentangle the nucleotide binding and the global conformational changes of the protein. This allows us to directly observe the cooperativity between the two nucleotide binding pockets of the Hsp90 dimer.@en

The molecular chaperone and heat-shock protein Hsp90 has become a central target in anti-cancer therapy. Nevertheless, the effect of Hsp90 inhibition is still not understood at the molecular level, preventing a truly rational drug design. Here we report on the effect of the most prominent drug candidates, namely, radicicol, geldanamycin, derivatives of purine, and novobiocin, on Hsp90′s characteristic conformational dynamics and the binding of three interaction partners. Unexpectedly, the global opening and closing transitions are hardly affected by Hsp90 inhibitors. Moreover, we find no significant changes in the binding of the cochaperones Aha1 and p23 nor of the model substrate Δ131Δ. This holds true for competitive and allosteric inhibitors. Therefore, direct inhibition mechanisms affecting only one molecular interaction are unlikely. We suggest that the inhibitory action observed in vivo is caused by a combination of subtle effects, which can be used in the search for novel Hsp90 inhibition mechanisms.@en

Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods.

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Single molecule time traces reveal the time evolution of unsynchronized kinetic systems. Especially single molecule Förster resonance energy transfer (smFRET) provides access to enzymatically important time scales, combined with molecular distance resolution and minimal interference with the sample. Yet the kinetic analysis of smFRET time traces is complicated by experimental shortcomings - such as photo-bleaching and noise. Here we recapitulate the fundamental limits of single molecule fluorescence that render the classic, dwell-time based kinetic analysis unsuitable. In contrast, our Single Molecule Analysis of Complex Kinetic Sequences (SMACKS) considers every data point and combines the information of many short traces in one global kinetic rate model. We demonstrate the potential of SMACKS by resolving the small kinetic effects caused by different ionic strengths in the chaperone protein Hsp90. These results show an unexpected interrelation between conformational dynamics and ATPase activity in Hsp90.

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