Substrate Activity Screening with Kinases: Discovery of Small‐Molecule Substrate‐Competitive c‐Src Inhibitors

Substrate‐competitive kinase inhibitors represent a promising class of kinase inhibitors, however, there is no methodology to selectively identify this type of inhibitor. Substrate activity screening was applied to tyrosine kinases. By using this methodology, the first small‐molecule substrates for any protein kinase were discovered, as well as the first substrate‐competitive inhibitors of c‐Src with activity in both biochemical and cellular assays. Characterization of the lead inhibitor demonstrates that substrate‐competitive kinase inhibitors possess unique properties, including cellular efficacy that matches biochemical potency and synergy with ATP‐competitive inhibitors.     

Misfolding of Luciferase at the Single‐Molecule Level

The folding of complex proteins can be dramatically affected by misfolding transitions. Directly observing misfolding and distinguishing it from aggregation is challenging. Experiments with optical tweezers revealed transitions between the folded states of a single protein in the absence of mechanical tension. Nonfolded chains of the multidomain protein luciferase folded within seconds to different partially folded states, one of which was stable over several minutes and was more resistant to forced unfolding than other partially folded states. Luciferase monomers can thus adopt a stable misfolded state and can do so without interacting with aggregation partners. This result supports the notion that luciferase misfolding is the cause of the low refolding yields and aggregation observed with this protein. This approach could be used to study misfolding transitions in other large proteins, as well as the factors that affect misfolding.     

Dissecting Cooperative Communications in a Protein with a High‐Throughput Single‐Molecule Scalpel

Miscued communication often leads to misfolding and aggregation of the proteins involved in many diseases. Owing to the ensemble average property of conventional techniques, detailed communication diagrams are difficult to obtain. Mechanical unfolding affords an unprecedented perspective on cooperative transitions by observing a protein along a trajectory defined by two mutated cysteine residues. Nevertheless, this approach requires tedious sample preparation at the risk of altering native protein conformations. To address these issues, we applied click chemistry to tether a protein to the two dsDNA handles through primary amines in lysine residues as well as at the N terminus. As a proof of concept, we used laser tweezers to mechanically unfold and refold calmodulin along 36 trajectories, maximally allowed by this strategy in a single batch of protein preparation. Without a priori knowledge of the particular residues to which the double‐stranded DNA handles attach, we used hierarchical cluster analysis to identify 20 major trajectories, according to the size and the pattern of unfolding transitions. We dissected the cooperativity into all‐or‐none and partially cooperative events, which represent strong and weak high‐order interactions in proteins, respectively. Although the overall cooperativity is higher within the N or C lobe than that between the lobes, the all‐or‐none cooperativity is anisotropic among different the unfolding trajectories and becomes relatively more predominant when the size of the protein segments increases. The average cooperativity for all‐or‐none transitions falls within the expected range observed by ensemble techniques, which supports the hypothesis that unfolding of a free protein can be reconstituted from individual trajectories.     

Discovery of Small‐Molecule Interleukin‐2 Inhibitors from a DNA‐Encoded Chemical Library

Libraries of chemical compounds individually coupled to encoding DNA tags (DNA‐encoded chemical libraries) hold promise to facilitate exceptionally efficient ligand discovery. We constructed a high‐quality DNA‐encoded chemical library comprising 30 000 drug‐like compounds; this was screened in 170 different affinity capture experiments. High‐throughput sequencing allowed the evaluation of 120 million DNA codes for a systematic analysis of selection strategies and statistically robust identification of binding molecules. Selections performed against the tumor‐associated antigen carbonic anhydrase IX (CA IX) and the pro‐inflammatory cytokine interleukin‐2 (IL‐2) yielded potent inhibitors with exquisite target specificity. The binding mode of the revealed pharmacophore against IL‐2 was confirmed by molecular docking. Our findings suggest that DNA‐encoded chemical libraries allow the facile identification of drug‐like ligands principally to any protein of choice, including molecules capable of disrupting high‐affinity protein–protein interactions.     

Single‐Molecule Electroluminescence of a Phosphorescent Organometallic Complex

Lighting one by one: The electroluminescence (EL) from single molecules of a red phosphorescent iridium complex dispersed in a hole‐transporting polymer matrix is studied. The single‐molecule EL dynamics is determined by local structural inhomogeneities in the matrix polymer (see picture).

     

Peering into Biological Nanopore: A Practical Technology to Single‐Molecule Analysis

As a unique technique at the singe‐molecule level to explore the distribution and temporal order of events, nanopore technology has attracted increasing attention. In comparison to the previous applications in DNA sequencing, this Focus Review highlights the technical details of biological nanopores, especially α‐hemolysin, in the analysis of peptides and proteins. The instrument configurations, experimental interferences, and data analysis including the conformation of peptides and proteins and their interactions for single‐molecule detection are discussed.     

Analysis of Single‐Molecule Fluorescence Spectroscopic Data with a Markov‐Modulated Poisson Process

We present a photon‐by‐photon analysis framework for the evaluation of data from single‐molecule fluorescence spectroscopy (SMFS) experiments using a Markov‐modulated Poisson process (MMPP). A MMPP combines a discrete (and hidden) Markov process with an additional Poisson process reflecting the observation of individual photons. The algorithmic framework is used to automatically analyze the dynamics of the complex formation and dissociation of Cu²⁺ ions with the bidentate ligand 2,2′‐bipyridine‐4,4′dicarboxylic acid in aqueous media. The process of association and dissociation of Cu²⁺ ions is monitored with SMFS. The dcbpy‐DNA conjugate can exist in two or more distinct states which influence the photon emission rates. The advantage of a photon‐by‐photon analysis is that no information is lost in preprocessing steps. Different model complexities are investigated in order to best describe the recorded data and to determine transition rates on a photon‐by‐photon basis. The main strength of the method is that it allows to detect intermittent phenomena which are masked by binning and that are difficult to find using correlation techniques when they are short‐lived.     

Identifying Multiple Populations from Single‐Molecule Lifetime Distributions

A major advantage of single‐molecule methods over ensemble‐averaging techniques involves the ability to characterize heterogeneity through the identification of multiple molecular populations. It can be challenging, however, to determine absolute values of dynamic parameters (and to relate these values to those determined from a conventional method) because characteristic timescales of various populations may vary over many orders of magnitude, and under a given set of experimental conditions instrumental sensitivity to various populations may be unequal. Using data obtained from the single‐molecule tracking microscopy of fibrinogen protein adsorption and desorption, it is shown that by performing a combined analysis of molecular trajectories obtained using a range of acquisition times, it is possible to extract quantitative absolute values of multiple population fractions and residence times (with well‐defined uncertainties), even when these values span many orders of magnitude. In particular, as many as six distinct populations are rigorously identified, exhibiting characteristic timescales that vary over nearly three orders of magnitude with population fractions as small as one part in a thousand. This approach will lead to better comparability between single‐molecule experiments and may be useful in connecting single‐molecule to ensemble‐averaged observations.     

Fast Biosynthesis of GFP Molecules: A Single‐Molecule Fluorescence Study

It's not easy being green : Real‐time visualization of labeled ribosomes and de novo synthesized green fluorescent protein molecules using single‐molecule‐sensitive fluorescence microscopy demonstrates that the mutant GFPem is produced with a characteristic time of five minutes. Fluorescence of the fastest GFP molecules appears within one minute (see picture).

     

Reversible Unfolding–Refolding of Rubredoxin: A Single‐Molecule Force Spectroscopy Study

In metalloproteins, metal centers serve as active sites for a range of functional purposes and as important structural elements to facilitate protein folding and assembly. It is challenging to observe the reversible unfolding and refolding of metalloproteins because of a loss or decomposition of the metal center. Here, the reversible unfolding–refolding of the iron–sulfur protein rubredoxin was observed directly using single‐molecule force spectroscopy. The results demonstrate that the iron can remain attached to the CXXC motif when rubredoxin is unfolded. Upon relaxation, the unfolded rubredoxin can refold into its native holo state with the reconstituted FeS₄ center. The possible loss of iron from the unfolded protein prevents rubredoxin from refolding into its native holo state. These results demonstrated that unfolding of rubredoxin is reversible, as long as the iron remains attached, and provide experimental evidence for the iron‐priming mechanism for the folding of rubredoxin.     

DCDHF Fluorophores for Single‐Molecule Imaging in Cells

There is a persistent need for small‐molecule fluorescent labels optimized for single‐molecule imaging in the cellular environment. Application of these labels comes with a set of strict requirements: strong absorption, efficient and stable emission, water solubility and membrane permeability, low background emission, and red‐shifted absorption to avoid cell autofluorescence. We have designed and characterized several fluorophores, termed “DCDHF” fluorophores, for use in live‐cell imaging based on the push–pull design: an amine donor group and a 2‐dicyanomethylene‐3‐cyano‐2,5‐dihydrofuran (DCDHF) acceptor group, separated by a π‐rich conjugated network. In general, the DCDHF fluorophores are comparatively photostable, sensitive to local environment, and their chemistries and photophysics are tunable to optimize absorption wavelength, membrane affinity, and solubility. Especially valuable are fluorophores with sophisticated photophysics for applications requiring additional facets of control, such as photoactivation. For example, we have reengineered a red‐emitting DCDHF fluorophore so that it is dark until photoactivated with a short burst of low‐intensity violet light. This molecule and its relatives provide a new class of bright photoactivatable small‐molecule fluorophores, which are needed for super‐resolution imaging schemes that require active control (here turning‐on) of single‐molecule emission.     

Characterizing Single‐Molecule FRET Dynamics with Probability Distribution Analysis

Probability distribution analysis (PDA) is a recently developed statistical tool for predicting the shapes of single‐molecule fluorescence resonance energy transfer (smFRET) histograms, which allows the identification of single or multiple static molecular species within a single histogram. We used a generalized PDA method to predict the shapes of FRET histograms for molecules interconverting dynamically between multiple states. This method is tested on a series of model systems, including both static DNA fragments and dynamic DNA hairpins. By fitting the shape of this expected distribution to experimental data, the timescale of hairpin conformational fluctuations can be recovered, in good agreement with earlier published results obtained using different techniques. This method is also applied to studying the conformational fluctuations in the unliganded Klenow fragment (KF) of Escherichia coli DNA polymerase I, which allows both confirmation of the consistency of a simple, two‐state kinetic model with the observed smFRET distribution of unliganded KF and extraction of a millisecond fluctuation timescale, in good agreement with rates reported elsewhere. We expect this method to be useful in extracting rates from processes exhibiting dynamic FRET, and in hypothesis‐testing models of conformational dynamics against experimental data.