Calmodulin is the primary calcium binding protein in living cells. Its function and structure depend strongly on calcium concentration. We used single molecule force spectroscopy by optical tweezers ...to study the folding of calmodulin in the physiologically relevant range. We find that full-length calmodulin switches from a rich and complex folding behavior at high calcium to a simple folding pathway at apo conditions. Using truncation mutants, we studied the individual domains separately. Folding and stability of the individual domains differ significantly at low calcium concentrations. With increasing calcium, the folding rate constants increase while unfolding rate constants decrease. The complete kinetic as well as energetic behavior of both domains could be modeled using a calcium-dependent three-pathway model. We find that the dominant folding pathway at high calcium concentrations proceeds via a transition state capable of binding one calcium ion. The folding of calmodulin seems to be designed to occur fast robustly over a large range of calcium concentrations and hence energetic stabilities.
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The importance of the protein corona formed around nanoparticles upon entering a biological fluid has recently been highlighted. This corona is, when sufficiently long-lived, thought to govern the ...particles’ biological fate. However, even this long-lived “hard” corona evolves and re-equilibrates as particles pass from one biological fluid to another, and may be an important feature for long-term fate. Here we show the evolution of the protein corona as a result of transfer of nanoparticles from one biological fluid (plasma) into another (cytosolic fluid), a simple illustrative model for the uptake of nanoparticles into cells. While no direct comparison can be made to what would happen in, for example, the uptake pathway, the results confirm that significant evolution of the corona occurs in the second biological solution, but that the final corona contains a “fingerprint” of its history. This could be evolved to map the transport pathways utilized by nanoparticles, and eventually to predict nanoparticle fate and behavior.
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IJS, KILJ, NUK, PNG, UL, UM
Nanoparticles in a biological fluid (plasma, or otherwise) associate with a range of biopolymers, especially proteins, organized into the "protein corona" that is associated with the nanoparticle and ...continuously exchanging with the proteins in the environment. Methodologies to determine the corona and to understand its dependence on nanomaterial properties are likely to become important in bionanoscience. Here, we study the long-lived ("hard") protein corona formed from human plasma for a range of nanoparticles that differ in surface properties and size. Six different polystyrene nanoparticles were studied: three different surface chemistries (plain PS, carboxyl-modified, and amine-modified) and two sizes of each (50 and 100 nm), enabling us to perform systematic studies of the effect of surface properties and size on the detailed protein coronas. Proteins in the corona that are conserved and unique across the nanoparticle types were identified and classified according to the protein functional properties. Remarkably, both size and surface properties were found to play a very significant role in determining the nanoparticle coronas on the different particles of identical materials. We comment on the future need for scientific understanding, characterization, and possibly some additional emphasis on standards for the surfaces of nanoparticles.
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Cohesin is essential for the hierarchical organization of the eukaryotic genome and plays key roles in many aspects of chromosome biology. The conformation of cohesin bound to DNA remains poorly ...defined, leaving crucial gaps in our understanding of how cohesin fulfills its biological functions. Here, we use single-molecule microscopy to directly observe the dynamic and functional characteristics of cohesin bound to DNA. We show that cohesin can undergo rapid one-dimensional (1D) diffusion along DNA, but individual nucleosomes, nucleosome arrays, and other protein obstacles significantly restrict its mobility. Furthermore, we demonstrate that DNA motor proteins can readily push cohesin along DNA, but they cannot pass through the interior of the cohesin ring. Together, our results reveal that DNA-bound cohesin has a central pore that is substantially smaller than anticipated. These findings have direct implications for understanding how cohesin and other SMC proteins interact with and distribute along chromatin.
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•Individual molecules of cohesin form topologically bound complexes that diffuse on DNA•Diffusing complexes show high mobility and long lifetimes on DNA•Bypassing of obstacles places limits on the size of the DNA-binding pore•Translocases can push cohesins along DNA
Stigler et al. study the loading and interactions of cohesin complexes with DNA and protein obstacles using single-molecule microscopy. Individual molecules of cohesin form topologically bound complexes that diffuse rapidly on DNA. Collisions with small obstacles reveal that the cohesin pore may be smaller than implied by prevailing models.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Direct observation of the detailed conformational fluctuations of a single protein molecule en route to its folded state has so far been realized only in silico. We have used single-molecule force ...spectroscopy to study the folding transitions of single calmodulin molecules. High-resolution optical tweezers assays in combination with hidden Markov analysis reveal a complex network of on-and off-pathway intermediates. Cooperative and anticooperative interactions across domain boundaries can be observed directly. The folding network involves four intermediates. Two off-pathway intermediates exhibit non-native interdomain interactions and compete with the ultrafast productive folding pathway.
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Mechanical forces are important signals for cell response and development, but detailed molecular mechanisms of force sensing are largely unexplored. The cytoskeletal protein filamin is a key ...connecting element between the cytoskeleton and transmembrane complexes such as integrins or the von Willebrand receptor glycoprotein Ib. Here, we show using single-molecule mechanical measurements that the recently reported Ig domain pair 20–21 of human filamin A acts as an autoinhibited force-activatable mechanosensor. We developed a mechanical single-molecule competition assay that allows online observation of binding events of target peptides in solution to the strained domain pair. We find that filamin force sensing is a highly dynamic process occurring in rapid equilibrium that increases the affinity to the target peptides by up to a factor of 17 between 2 and 5 pN. The equilibrium mechanism we find here can offer a general scheme for cellular force sensing.
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Protein folding is governed by non-covalent interactions under the benefits and constraints of the covalent linkage of the backbone chain. In the current work we investigate the influence of loop ...length variation on the free energies of folding and ligand binding in a small globular single-domain protein containing two EF-hand subdomains-calbindin D
. We introduce a linker extension between the subdomains and vary its length between 1 to 16 glycine residues. We find a close to linear relationship between the linker length and the free energy of folding of the Ca
-free protein. In contrast, the linker length has only a marginal effect on the Ca
affinity and cooperativity. The variant with a single-glycine extension displays slightly increased Ca
affinity, suggesting that the slightly extended linker allows optimized packing of the Ca
-bound state. For the extreme case of disconnected subdomains, Ca
binding becomes coupled to folding and assembly. Still, a high affinity between the EF-hands causes the non-covalent pair to retain a relatively high apparent Ca
affinity. Our results imply that loop length variation could be an evolutionary option for modulating properties such as protein stability and turnover without compromising the energetics of the specific function of the protein.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Force-spectroscopic measurements of ligand-receptor systems and the unfolding/folding of nucleic acids or proteins reveal information on the underlying energy landscape along the pulling coordinate. ...The slope Δx‡ of the force-dependent unfolding/unbinding rates is interpreted as the distance from the folded/bound state to the transition state for unfolding/unbinding and, hence, often related to the mechanical compliance of the sample molecule. Here we show that in ligand-binding proteins, the experimentally inferred Δx‡ can depend on the ligand concentration, unrelated to changes in mechanical compliance. We describe the effect in single-molecule, force-spectroscopy experiments of the calcium-binding protein calmodulin and explain it in a simple model where mechanical unfolding and ligand binding occur on orthogonal reaction coordinates. This model predicts changes in the experimentally inferred Δx‡, depending on ligand concentration and the associated shift of the dominant barrier between the two reaction coordinates. We demonstrate quantitative agreement between experiments and simulations using a realistic six-state kinetic scheme using literature values for calcium-binding kinetics and affinities. Our results have important consequences for the interpretation of force-spectroscopic data of ligand-binding proteins.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
In this study we expand the accessible dynamic range of single-molecule force spectroscopy by optical tweezers to the microsecond range by fast sampling. We are able to investigate a single molecule ...for up to 15 min and with 300-kHz bandwidth as the protein undergoes tens of millions of folding/unfolding transitions. Using equilibrium analysis and autocorrelation analysis of the time traces, the full energetics as well as real-time kinetics of the ultrafast folding of villin headpiece 35 and a stable asparagine 68 alanine/lysine 70 methionine variant can be measured directly. We also performed Brownian dynamics simulations of the response of the bead-DNA system to protein-folding fluctuations. All key features of the force-dependent deflection fluctuations could be reproduced: SD, skewness, and autocorrelation function. Our measurements reveal a difference in folding pathway and cooperativity between wild-type and stable variant of headpiece 35. Autocorrelation force spectroscopy pushes the time resolution of single-molecule force spectroscopy to ∼10 µs thus approaching the timescales accessible for all atom molecular dynamics simulations.
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The ever more complex fluctuation patterns discovered by single molecule experiments require statistical methods to analyze multi‐state hopping traces of long lengths. Hidden Markov modeling is a ...statistical tool that offers the scalability to analyze even complex data and extract kinetic information. We give an introduction on how to implement hidden Markov modeling for the analysis of single molecule force spectroscopic traces, deal with missed events, and test the method on a calcium binding protein.
Complex fluctuation patterns discovered by single‐molecule experiments require statistical methods to analyze long multistate hopping traces. The authors give an introduction on how to implement hidden Markov modeling for this purpose and test the method on a calcium binding protein.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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