Individual Protein Molecules Research Articles

Single-Molecule Force Spectroscopy Studies on Sumo Polyproteins Reveal their Mechanical Flexibility

Small ubiquitin-like modifiers (SUMOs) are implicated in regulating protein function through post-translational modification that is akin to ubiquitin. Ubiquitin and SUMO proteins belong to β-grasp topology and share >95% structural homology. Similar to ubiquitin, SUMOs are also found to form polymers in vitro and in vivo. However, the functional significance of polySUMOs remains to be understood. We have used protein engineering to construct polyproteins, (SUMO1)8 and (SUMO2)8, in which individual protein molecules are linked in tandem through N-C termini with peptide linkages. Mechanical properties of these polyproteins are measured using single-molecule force spectroscopy and compared them with those of polyubiquitins. We observed a two-state mechanical unfolding pathway for SUMO1 and SUMO2, which is similar to that of ubiquitin. Nevertheless, the unfolding forces of SUMO1 (∼130 pN) and SUMO2 (∼120 pN) are lower than that of ubiquitin (∼190 pN) indicating their lower mechanical stability. The mechanical stabilities of SUMO proteins and ubiquitin are well correlated with the number of inter-residue contacts present in their structures. From pulling speed dependent mechanical unfolding experiments and Monte Carlo simulations, we find that the unfolding potential widths of SUMO1 (∼0.51 nm) and SUMO2 (∼0.33 nm) are much larger than that of ubiquitin (∼0.19 nm), indicating that SUMO1 is 6 times and SUMO2 is 3 times more mechanically flexible than ubiquitin. Interestingly, SUMO polyproteins are mechanically stronger than the physiologically relevant Lys48-C-linked polyubiquitin, which unfolds at very low unfolding forces ∼85 pN. This might suggest a possible mechanical role of polySUMOs in proteasomal degradation, which is in concurrence with the recent studies showing crosstalk between polySUMOs and polyubiquitin in targeting proteins for degradation. We conclude that the mechanical flexibility of SUMOs might also have implications in modulating the function of target proteins through SUMOylation.

Selective fluorescent-free detection of biomolecules on nanobiochips by wavelength dependent-enhanced dark field illumination

Individual silver nanoparticle-conjugated target protein (cTnI) molecules on gold-nanopatterned chip were selectively detected by wavelength dependent-enhanced dark field illumination. Using specific nanoparticles with unique sizes and materials, the immunotargeted nanoparticle on the chips was detected at the single-molecule level by monitoring changes in the plasmonic resonance based on wavelength dependence.

Filming Dynamic Processes of Proteins by High-Speed Afm

Directly observing individual protein molecules in action at high spatiotemporal resolution has long been the holy grail for biological science. High-speed atomic force microscopy (HS-AFM) which can perform this type of observations is now materialized. It opens up a new opportunity to directly observe the structure dynamics and dynamic processes of biological molecules in physiological solutions, at subsecond to sub-100 ms temporal resolution and ∼2 nm lateral and 0.1 nm vertical spatial resolution. Importantly, the tip-sample interaction does not disturb their function. In fact, functioning molecules, such as myosin V walking on an actin filament and bacteriorhodopsin in response to light, have been successfully visualized. In the quest for the functional mechanism of proteins, inferences no longer have to be made from static snapshots of their molecular structures and dynamic behavior of optical markers attached to proteins. High-resolution molecular movies reveal the details of dynamic behavior of molecules in action, which can be interpreted without intricate analyses and interpretations. In this talk, I will highlight recent imaging studies and then describe the fundamentals behind the achieved high imaging rate and low-invasiveness to the sample.

T4 Lysozyme as a Pac-Man: How Fast Can It Chew?

T4 Lysozyme as a Pac-Man: How Fast Can It Chew?

Phosphoproteomics and Lung Cancer Research

Massive evidence suggests that genetic abnormalities contribute to the development of lung cancer. These molecular abnormalities may serve as diagnostic, prognostic and predictive biomarkers for this deadly disease. It is imperative to search these biomarkers in different tumorigenesis pathways so as to provide the most appropriate therapy for each individual patient with lung malignancy. Phosphoproteomics is a promising technology for the identification of biomarkers and novel therapeutic targets for cancer. Thousands of proteins interact via physical and chemical association. Moreover, some proteins can covalently modify other proteins post-translationally. These post-translational modifications ultimately give rise to the emergent functions of cells in sequence, space and time. Phosphoproteomics clinical researches imply the comprehensive analysis of the proteins that are expressed in cells or tissues and can be employed at different stages. In addition, understanding the functions of phosphorylated proteins requires the study of proteomes as linked systems rather than collections of individual protein molecules. In fact, proteomics approaches coupled with affinity chromatography strategies followed by mass spectrometry have been used to elucidate relevant biological questions. This article will discuss the relevant clues of post-translational modifications, phosphorylated proteins, and useful proteomics approaches to identify molecular cancer signatures. The recent progress in phosphoproteomics research in lung cancer will be also discussed.

Silica as a matrix for encapsulating proteins: surface effects on protein structure assessed by circular dichroism spectroscopy.

The encapsulation of biomolecules in solid materials that retain the native properties of the molecule is a desired feature for the development of biosensors and biocatalysts. In the current study, protein entrapment in silica-based materials is explored using the sol-gel technique. This work surveys the effects of silica confinement on the structure of several model polypeptides, including apomyoglobin, copper-zinc superoxide dismutase, polyglutamine, polylysine, and type I antifreeze protein. Changes in the secondary structure of each protein following encapsulation are monitored by circular dichroism spectroscopy. In many cases, silica confinement reduces the fraction of properly-folded protein relative to solution, but addition of a secondary solute or modification of the silica surface leads to an increase in structure. Refinement of the glass surface by addition of a monosubstituted alkoxysilane during sol-gel processing is shown to be a valuable tool for testing the effects of surface chemistry on protein structure. Because silica entrapment prevents protein aggregation by isolating individual protein molecules in the pores of the glass material, one may monitor aggregation-prone polypeptides under solvent conditions that are prohibited in solution, as demonstrated with polyglutamine and a disease-related variant of superoxide dismutase.

Release pathways of interferon α2a molecules from lipid twin screw extrudates revealed by single molecule fluorescence microscopy

The pathways of interferon α2a release from a triglyceride based implant system were studied by single molecule fluorescence microscopy. The protein was labeled with a stable fluorescent dye ATTO647N, freeze-dried and embedded into the lipid matrix via twin-screw extrusion. The implant system consisted of a pore-forming agent (water soluble PEG 6000) and two types of triglycerides with different melting ranges which allowed the production of the implants at moderate temperatures and without the use of organic solvents. Single molecule microscopy and single particle tracking of labeled proteins contained in these implants revealed that two populations of diffusing proteins were present. Moreover, proteins were not only released via water-filled pores (created by dissolution of the pore-former), but surprisingly also through diffusion in a phase of molten lipid. Diffusion coefficients of IFNα 2a derived by tracking of individual protein molecules within the implant system were similar to diffusion coefficients obtained from control measurements in pure molten lipid and highly concentrated solutions of PEG 6000. In conclusion, tracking of individual protein molecules was successfully used to elucidate the release pathways of proteins from a relevant lipid based implant system.

Two Dimensional Nanoarrays of Individual Protein Molecules

Protein molecules on solid surfaces are essential to a number of applications, such as biosensors, biomaterials, and drug delivery. In most approaches for protein immobilization, inter-molecular distances on the solid surface are not controlled and this may lead to aggregation and crowding. Here, a simple approach to immobilize individual protein molecules in a well-ordered 2D array is shown, using nanopatterns obtained from a polystyrene-block-poly(2-hydroxyethyl methacrylate) (PS-b-PHEMA) diblock copolymer thin film. This water-stable and protein-resistant polymer film contains hexagonally ordered PS cylindrical domains in a PHEMA matrix. The PS domains are activated by incorporating alkyne-functionalized PS and immobilizing azide-tagged proteins specifically onto each PS domain using "Click" chemistry. The nanometer size of the PS domain dictates that each domain can accommodate no more than one protein molecule, as verified by atomic force microscopy imaging. Immunoassay shows that the amount of specifically bound antibody scales with the number density of individual protein molecules on the 2D nanoarrays.

Characterization of the motion of membrane proteins using high-speed atomic force microscopy

For cells to function properly, membrane proteins must be able to diffuse within biological membranes. The functions of these membrane proteins depend on their position and also on protein-protein and protein-lipid interactions. However, so far, it has not been possible to study simultaneously the structure and dynamics of biological membranes. Here, we show that the motion of unlabelled membrane proteins can be characterized using high-speed atomic force microscopy. We find that the molecules of outer membrane protein F (OmpF) are widely distributed in the membrane as a result of diffusion-limited aggregation, and while the overall protein motion scales roughly with the local density of proteins in the membrane, individual protein molecules can also diffuse freely or become trapped by protein-protein interactions. Using these measurements, and the results of molecular dynamics simulations, we determine an interaction potential map and an interaction pathway for a membrane protein, which should provide new insights into the connection between the structures of individual proteins and the structures and dynamics of supramolecular membranes.

In Situ Observation of Elementary Growth Processes of Protein Crystals by Advanced Optical Microscopy

To start systematically investigating the quality improvement of protein crystals, the elementary growth processes of protein crystals must be first clarified comprehensively. Atomic force microscopy (AFM) has made a tremendous contribution toward elucidating the elementary growth processes of protein crystals and has confirmed that protein crystals grow layer by layer utilizing kinks on steps, as in the case of inorganic and low-molecular-weight compound crystals. However, the scanning of the AFM cantilever greatly disturbs the concentration distribution and solution flow in the vicinity of growing protein crystals. AFM also cannot visualize the dynamic behavior of mobile solute and impurity molecules on protein crystal surfaces. To compensate for these disadvantages of AFM, in situ observation by two types of advanced optical microscopy has been recently performed. To observe the elementary steps of protein crystals noninvasively, laser confocal microscopy combined with differential interference contrast microscopy (LCM-DIM) was developed. To visualize individual mobile protein molecules, total internal reflection fluorescent (TIRF) microscopy, which is widely used in the field of biological physics, was applied to the visualization of protein crystal surfaces. In this review, recent progress in the noninvasive in situ observation of elementary steps and individual mobile protein molecules on protein crystal surfaces is outlined.

A Close Look at Proteins: Submolecular Resolution of Two- and Three-Dimensionally Folded Cytochrome c at Surfaces

Imaging of individual protein molecules at the single amino acid level has so far not been possible due to the incompatibility of proteins with the vacuum environment necessary for high-resolution scanning probe microscopy. Here we demonstrate electrospray ion beam deposition of selectively folded and unfolded cytochrome c protein ions on atomically defined solid surfaces in ultrahigh vacuum (10(-10) mbar) and achieve unprecedented resolution with scanning tunneling microscopy. On the surface folded proteins are found to retain their three-dimensional structure. Unfolded proteins are observed as extended polymer strands displaying submolecular features with resolution at the amino acid level. On weakly interacting surfaces, unfolded proteins refold into flat, irregular patches composed of individual molecules. This suggests the possibility of two-dimensionally confined folding of peptides of an appropriate sequence into regular two-dimensional structures as a new approach toward functional molecular surface coatings.

Contributions of fluorescence techniques to understanding G protein-coupled receptor dimerisation.

G protein-coupled receptors (GPCRs) are the largest class of eukaryotic cell-surface receptors and, over the last decade, it has become clear that they are capable of dimerisation. Whilst many biochemical and biophysical approaches have been used to study dimerisation, fluorescence techniques, including Förster resonance energy transfer and single molecule fluorescence, have been key players. Here we review recent contributions of fluorescence techniques to investigate GPCR dimers, including dimerisation in cell membranes and native tissues, the effect of ligand binding on dimerisation and the kinetics of dimer formation and dissociation. The challenges of studying multicomponent membrane protein systems have led to the development and refinement of many fluorescence assays, allowing the functional consequences of receptor dimerisation to be investigated and individual protein molecules to be imaged in the membranes of living cells. It is likely that the fluorescence techniques described here will be of use for investigating many other multicomponent membrane protein systems.

Relevant phosphoproteomic and mass spectrometry: approaches useful in clinical research

Background"It's not what we do, it's the way that we do it". Never has this maxim been truer in proteomics than now. Mass Spectrometry-based proteomics/phosphoproteomics tools are critical to understand the structure and dynamics (spatial and temporal) of signalling that engages and migrates through the entire proteome. Approaches such as affinity purification followed by Mass Spectrometry (MS) have been used to elucidate relevant biological questions disease vs. health. Thousands of proteins interact via physical and chemical association. Moreover, certain proteins can covalently modify other proteins post-translationally. These post-translational modifications (PTMs) ultimately give rise to the emergent functions of cells in sequence, space and time.FindingsUnderstanding the functions of phosphorylated proteins thus requires one to study proteomes as linked-systems rather than collections of individual protein molecules. Indeed, the interacting proteome or protein-network knowledge has recently received much attention, as network-systems (signalling pathways) are effective snapshots in time, of the proteome as a whole. MS approaches are clearly essential, in spite of the difficulties of some low abundance proteins for future clinical advances.ConclusionClinical proteomics-MS has come a long way in the past decade in terms of technology/platform development, protein chemistry, and together with bioinformatics and other OMICS tools to identify molecular signatures of diseases based on protein pathways and signalling cascades. Hence, there is great promise for disease diagnosis, prognosis, and prediction of therapeutic outcome on an individualized basis. However, and as a general rule, without correct study design, strategy and implementation of robust analytical methodologies, the efforts, efficiency and expectations to make biomarkers (especially phosphorylated kinases) a useful reality in the near future, can easily be hampered.

A Hybrid Tirf-Magnetic Tweezers Instrument for Studying Sub-Nanometer Effects of Force on Proteins

Proteins exert and withstand mechanical force in many fundamental biological processes. Optical tweezers have become a useful research tool for applying forces to single proteins and measuring the resulting changes in extension, but many interesting processes produce changes smaller than their resolution limit. Our experimental setup skirts this limitation by measuring distance changes using single-molecule Forster resonance energy transfer (smFRET) produced from a total internal reflection fluorescence (TIRF) microscope incorporating magnetic tweezers. Individual protein molecules are conjugated to FRET-paired fluorescent dyes and functionalized DNA handles using maleimide and click chemistry. These handles tether each molecule between a glass coverslip within the TIRF microscope and a paramagnetic bead. An external magnet applies a uniform field that exerts a force on each molecule tethered to the surface. Because the FRET from each molecule in the microscope's field of view can be measured simultaneously, the extension between dyes of many individual molecules as a function of force can be monitored in parallel. Here we present our initial studies using this new setup.

Characterization of Glucocorticoid Receptor-hsp90 Chaperone Machinery Heterocomplex Assembly using Atomic Force Microscopy

Atomic force microscopy (AFM) has demonstrated ability to provide direct visualization of individual molecules of proteins as well as multimeric protein-protein and DNA-protein complexes in near real-time and in buffered solutions. Here, we use tapping-mode AFM to visually analyze the interaction of the essential eukaryotic molecular chaperone protein hsp90 with the glucocorticoid receptor (GR), a well-established hsp90 client protein. An issue of debate within the hsp90 community has been to what extent hsp90 works independently of other molecular chaperones, such as hsp70, or as part of a multiprotein machinery.

AFM-Enhanced Single-Molecule Spectroscopy Studies of Intermittent Coherence and Time Bunching Effect of Enzyme Dynamics

Enzymatic reactions are traditionally studied at the ensemble level, despite significant static and dynamic inhomogeneities. Subtle conformational changes play a crucial role in protein functions, especially in enzymatic reactions involving complex substrate-enzyme interactions and chemical reactions. We applied AFM-enhanced single-molecule spectroscopy to study the mechanisms and dynamics of enzymatic reactions involved with kinase and lysozyme proteins. Enzymatic reaction turnovers and the associated structure changes of individual protein molecules were observed simultaneously in real-time by single-molecule FRET detections. Our single-molecule spectroscopy measurements of enzymatic conformational dynamics have revealed time bunching effect and intermittent coherence in conformational state change dynamics involving in enzymatic reaction cycles. The coherent conformational state dynamics suggests that the enzymatic catalysis involves a multi-step conformational motion along the coordinates of substrate-enzyme complex formation and product releasing, presenting as an extreme dynamic behavior intrinsically related to the time bunching effect that we have reported previously. Our results support a multiple-conformational state model, being consistent with a complementary conformation selection and induced-fit enzymatic loop-gated conformational change mechanism in substrate-enzyme active complex formation. Our new approach is applicable to a wide range of single-molecule FRET measurements for protein conformational changes under enzymatic reactions.

Important Clues of Current Phosphoproteomic Approaches for Clinical Research

Mass Spectrometry-based phosphoproteomics tools are critical to understand the structure and dynamics of signalling that engages and migrates through the entire proteome. Approaches such as affinity purification followed by Mass Spectrometry (MS) have been used to elucidate relevant biological questions in health and disease. Thousands of proteins interact via physical and chemical association. Certain proteins can covalently modify other proteins post-translationally. These post-translational modifications (PTMs) ultimately give rise to the emergent functions of cells in sequence, space and time. Understanding the functions of phosphorylated proteins thus requires one to study proteomes as linked-systems rather than collections of individual protein molecules. The interacting proteome or protein-network knowledge has recently received much attention, as networksystems (signalling pathways) are effective snapshots in time of the proteome as a whole. MS approaches are clearly essential, in spite of the difficulties of some low abundance proteins (e.g some kinases).

Single-molecule protein arrays enabled by scanning probe block copolymer lithography

The ability to control the placement of individual protein molecules on surfaces could enable advances in a wide range of areas, from the development of nanoscale biomolecular devices to fundamental studies in cell biology. Such control, however, remains a challenge in nanobiotechnology due to the limitations of current lithographic techniques. Herein we report an approach that combines scanning probe block copolymer lithography with site-selective immobilization strategies to create arrays of proteins down to the single-molecule level with arbitrary pattern control. Scanning probe block copolymer lithography was used to synthesize individual sub-10-nm single crystal gold nanoparticles that can act as scaffolds for the adsorption of functionalized alkylthiol monolayers, which facilitate the immobilization of specific proteins. The number of protein molecules that adsorb onto the nanoparticles is dependent upon particle size; when the particle size approaches the dimensions of a protein molecule, each particle can support a single protein. This was demonstrated with both gold nanoparticle and quantum dot labeling coupled with transmission electron microscopy imaging experiments. The immobilized proteins remain bioactive, as evidenced by enzymatic assays and antigen-antibody binding experiments. Importantly, this approach to generate single-biomolecule arrays is, in principle, applicable to many parallelized cantilever and cantilever-free scanning probe molecular printing methods.

Ultrasensitive detection of protein translocated through toxin pores in droplet-interface bilayers

Many bacterial toxins form proteinaceous pores that facilitate the translocation of soluble effector proteins across cellular membranes. With anthrax toxin this process may be monitored in real time by electrophysiology, where fluctuations in ionic current through these pores inserted in model membranes are used to infer the translocation of individual protein molecules. However, detecting the minute quantities of translocated proteins has been a challenge. Here, we describe use of the droplet-interface bilayer system to follow the movement of proteins across a model membrane separating two submicroliter aqueous droplets. We report the capture and subsequent direct detection of as few as 100 protein molecules that have translocated through anthrax toxin pores. The droplet-interface bilayer system offers new avenues of approach to the study of protein translocation.

Direct Quantification of the Attempt Frequency Determining the Mechanical Unfolding of Ubiquitin Protein

Understanding protein dynamics requires a comprehensive knowledge of the underlying potential energy surface that governs the motion of each individual protein molecule. Single molecule mechanical studies have provided the unprecedented opportunity to study the individual unfolding pathways along a well defined coordinate, the end-to-end length of the protein. In these experiments, unfolding requires surmounting an energy barrier that separates the native from the extended state. The calculation of the absolute value of the barrier height has traditionally relied on the assumption of an attempt frequency, υ(‡). Here we used single molecule force-clamp spectroscopy to directly determine the value of υ(‡) for mechanical unfolding by measuring the unfolding rate of the small protein ubiquitin at varying temperatures. Our experiments demonstrate a significant effect of the temperature on the mechanical rate of unfolding. By extrapolating the unfolding rate in the absence of force for different temperatures, varying within the range spanning from 5 to 45 °C, we measured a value for the activation barrier of ΔG(‡) = 71 ± 5 kJ/mol and an exponential prefactor υ(‡) ∼4 × 10(9) s(-1). Although the measured prefactor value is 3 orders of magnitude smaller than the value predicted by the transition state theory (∼6 × 10(12) s(-1)), it is 400-fold higher than that encountered in analogous experiments studying the effect of temperature on the reactivity of a protein-embedded disulfide bond (∼10(7) M(-1) s(-1)). This approach will allow quantitative characterization of the complete energy landscape of a folding polypeptide from highly extended states, of capital importance for proteins with elastic function.