Nanometer-accuracy distance measurements between fluorophores at the single-molecule level.

Light microscopy is a powerful tool for probing the conformations of molecular machines at the single-molecule level. Single-molecule Förster resonance energy transfer can measure intramolecular distance changes of single molecules in the range of 2 to 8 nm. However, current superresolution measurements become error-prone below 25 nm. Thus, new single-molecule methods are needed for measuring distances in the 8- to 25-nm range. Here, we describe methods that utilize information about localization and imaging errors to measure distances between two different color fluorophores with ∼1-nm accuracy at distances >2 nm. These techniques can be implemented in high throughput using a standard total internal reflection fluorescence microscope and open-source software. We applied our two-color localization method to uncover an unexpected ∼4-nm nucleotide-dependent conformational change in the coiled-coil "stalk" of the motor protein dynein. We anticipate that these methods will be useful for high-accuracy distance measurements of single molecules over a wide range of length scales.

Structural insights into novel mechanisms of inhibition of the major ß-carbonic anhydrase CafB from the pathogenic fungus Aspergillus fumigatus.

In fungi the ß-class of carbonic anhydrases (ß-CAs) are zinc metalloenzymes that are essential for growth, survival, differentiation, and virulence. Aspergillus fumigatus is the most important pathogen responsible for invasive aspergillosis and possesses two major ß-CAs, CafA and CafB. Recently we reported the biochemical characterization and 1.8 Å crystal structure of CafA. Here, we report a crystallographic analysis of CafB revealing the mechanism of enzyme catalysis and establish the relationship of this enzyme to other ß-CAs. While CafA has a typical open conformation, CafB, when exposed to acidic pH and/or an oxidative environment, has a novel type of active site in which a disulfide bond is formed between two zinc-ligating cysteines, expelling the zinc ion and stabilizing the inactive form of the enzyme. Based on the structural data, we generated an oxidation-resistant mutant (Y159A) of CafB. The crystal structure of the mutant under reducing conditions retains a catalytic zinc at the expected position, tetrahedrally coordinated by three residues (C57, H113 and C116) and an aspartic acid (D59), and replacing the zinc-bound water molecule in the closed form. Furthermore, the active site of CafB crystals grown under zinc-limiting conditions has a novel conformation in which the solvent-exposed catalytic cysteine (C116) is flipped out of the metal coordination sphere, facilitating release of the zinc ion. Taken together, our results suggest that A. fumigatus use sophisticated activity-inhibiting strategies to enhance its survival during infection.

The structural basis of the low catalytic activities of the two minor ß-carbonic anhydrases of the filamentous fungus Aspergillus fumigatus.

The ß-carbonic anhydrases (ß-CAs) are widely distributed zinc-metalloenzymes that play essential roles in growth, survival, development and virulence in fungi. The majority of filamentous ascomycetes possess multiple ß-CA isoforms among which major and minor forms have been characterized. We examined the catalytic behavior of the two minor ß-CAs, CafC and CafD, of Aspergillus fumigatus, and found that both enzymes exhibited low CO2 hydration activities. To understand the structural basis of their low activities, we performed X-ray crystallographic and site-directed mutagenesis studies. Both enzymes exist as homodimers. Like other Type-I ß-CAs, the CafC active site has an "open" conformation in which the zinc ion is tetrahedrally coordinated by three residues (C36, H88 and C91) and a water molecule. However, L25 and L78 on the rim of the catalytic entry site protrude into the active site cleft, partially occluding access to it. Single (L25G or L78G) and double mutants provided evidence that widening the entrance to the active site greatly accelerates catalytic activity. By contrast, CafD has a typical Type-II "closed" conformation in which the zinc-bound water molecule is replaced by aspartic acid (D36). The most likely explanation for this result is that an arginine that is largely conserved within the ß-CA family is replaced by glycine (G38), so that D36 cannot undergo a conformational change by forming a D-R pair that creates the space for a zinc-bound water molecule and switches the enzyme to the active form. The CafD structure also reveals the presence of a "non-catalytic" zinc ion in the dimer interface, which may contribute to stabilizing the dimeric assembly.

Effects of hypertrophic and dilated cardiomyopathy mutations on power output by human ß-cardiac myosin.

Hypertrophic cardiomyopathy is the most frequently occurring inherited cardiovascular disease, with a prevalence of more than one in 500 individuals worldwide. Genetically acquired dilated cardiomyopathy is a related disease that is less prevalent. Both are caused by mutations in the genes encoding the fundamental force-generating protein machinery of the cardiac muscle sarcomere, including human ß-cardiac myosin, the motor protein that powers ventricular contraction. Despite numerous studies, most performed with non-human or non-cardiac myosin, there is no clear consensus about the mechanism of action of these mutations on the function of human ß-cardiac myosin. We are using a recombinantly expressed human ß-cardiac myosin motor domain along with conventional and new methodologies to characterize the forces and velocities of the mutant myosins compared with wild type. Our studies are extending beyond myosin interactions with pure actin filaments to include the interaction of myosin with regulated actin filaments containing tropomyosin and troponin, the roles of regulatory light chain phosphorylation on the functions of the system, and the possible roles of myosin binding protein-C and titin, important regulatory components of both cardiac and skeletal muscles.

Molecular consequences of the R453C hypertrophic cardiomyopathy mutation on human ß-cardiac myosin motor function.

Cardiovascular disorders are the leading cause of morbidity and mortality in the developed world, and hypertrophic cardiomyopathy (HCM) is among the most frequently occurring inherited cardiac disorders. HCM is caused by mutations in the genes encoding the fundamental force-generating machinery of the cardiac muscle, including ß-cardiac myosin. Here, we present a biomechanical analysis of the HCM-causing mutation, R453C, in the context of human ß-cardiac myosin. We found that this mutation causes a ∼30% decrease in the maximum ATPase of the human ß-cardiac subfragment 1, the motor domain of myosin, and a similar percent decrease in the in vitro velocity. The major change in the R453C human ß-cardiac subfragment 1 is a 50% increase in the intrinsic force of the motor compared with wild type, with no appreciable change in the stroke size, as observed with a dual-beam optical trap. These results predict that the overall force of the ensemble of myosin molecules in the muscle should be higher in the R453C mutant compared with wild type. Loaded in vitro motility assay confirms that the net force in the ensemble is indeed increased. Overall, this study suggests that the R453C mutation should result in a hypercontractile state in the heart muscle.

Single-molecule analysis of specificity and multivalency in binding of short linear substrate motifs to the APC/C.

Robust regulatory signals in the cell often depend on interactions between short linear motifs (SLiMs) and globular proteins. Many of these interactions are poorly characterized because the binding proteins cannot be produced in the amounts needed for traditional methods. To address this problem, we developed a single-molecule off-rate (SMOR) assay based on microscopy of fluorescent ligand binding to immobilized protein partners. We used it to characterize substrate binding to the Anaphase-Promoting Complex/Cyclosome (APC/C), a ubiquitin ligase that triggers chromosome segregation. We find that SLiMs in APC/C substrates (the D box and KEN box) display distinct affinities and specificities for the substrate-binding subunits of the APC/C, and we show that multiple SLiMs in a substrate generate a high-affinity multivalent interaction. The remarkably adaptable substrate-binding mechanisms of the APC/C have the potential to govern the order of substrate destruction in mitosis.

Crystal Structure of ß-Carbonic Anhydrase CafA from the Fungal Pathogen Aspergillus fumigatus.

The ß-class of carbonic anhydrases (ß-CAs) are zinc metalloenzymes widely distributed in the fungal kingdom that play essential roles in growth, survival, differentiation, and virulence by catalyzing the reversible interconversion of carbon dioxide (CO2) and bicarbonate (HCO3-). Herein, we report the biochemical and crystallographic characterization of the ß-CA CafA from the fungal pathogen Aspergillus fumigatus, the main causative agent of invasive aspergillosis. CafA exhibited apparent in vitro CO2 hydration activity in neutral to weak alkaline conditions, but little activity at acidic pH. The high-resolution crystal structure of CafA revealed a tetramer comprising a dimer of dimers, in which the catalytic zinc ion is tetrahedrally coordinated by three conserved residues (C119, H175, C178) and an acetate anion presumably acquired from the crystallization solution, indicating a freely accessible ″open″ conformation. Furthermore, knowledge of the structure of CafA in complex with the potent inhibitor acetazolamide, together with its functional intolerance of nitrate (NO3-) ions, could be exploited to develop new antifungal agents for the treatment of invasive aspergillosis.

Crystal Structure of a Highly Thermostable α-Carbonic Anhydrase from Persephonella marina EX-H1.

Bacterial α-type carbonic anhydrase (α-CA) is a zinc metalloenzyme that catalyzes the reversible and extremely rapid interconversion of carbon dioxide to bicarbonate. In this study, we report the first crystal structure of a hyperthermostable α-CA from Persephonella marina EXH1 (pm CA) in the absence and presence of competitive inhibitor, acetazolamide. The structure reveals a compactly folded pm CA homodimer in which each monomer consists of a 10-stranded ß-sheet in the center. The catalytic zinc ion is coordinated by three highly conserved histidine residues with an exchangeable fourth ligand (a water molecule, a bicarbonate anion, or the sulfonamide group of acetazolamide). Together with an intramolecular disulfide bond, extensive interfacial networks of hydrogen bonds, ionic and hydrophobic interactions stabilize the dimeric structure and are likely responsible for the high thermal stability. We also identified novel binding sites for calcium ions at the crystallographic interface, which serve as molecular glue linking negatively charged and otherwise repulsive surfaces. Furthermore, this large negatively charged patch appears to further increase the thermostability at alkaline pH range via favorable charge-charge interactions between pm CA and solvent molecules. These findings may assist development of novel α-CAs with improved thermal and/or alkaline stability for applications such as CO2 capture and sequestration.

Effect of surface undulation on polymer adsorption.

We study the adsorption of a long, flexible polymer (ideal or self-avoiding chain) interacting with a rough surface via a finite-range attraction. Within the Edwards equation approach, we develop a variational method to find the segmental distribution and the free energy of an adsorbed chain. As adsorption becomes strong, the segments tend to be localized within the valleys rather than above the hills of the undulating surface, resulting in a decrease of adsorption thickness. Consequently, the surface undulation enhances adsorption in the case of a strongly adsorbed chain whereas the undulation suppresses it for a weakly adsorbed one, since the enhanced entropic repulsion is dominant over the attraction from the surface. Considering the surface with undulation characterized by a Gaussian correlation as an example, we find an optimal correlation length at which the adsorption becomes the strongest and a critical correlation length below which desorption is induced.

Optimized measurements of separations and angles between intra-molecular fluorescent markers.

We demonstrate a novel, yet simple tool for the study of structure and function of biomolecules by extending two-colour co-localization microscopy to fluorescent molecules with fixed orientations and in intra-molecular proximity. From each colour-separated microscope image in a time-lapse movie and using only simple means, we simultaneously determine both the relative (x,y)-separation of the fluorophores and their individual orientations in space with accuracy and precision. The positions and orientations of two domains of the same molecule are thus time-resolved. Using short double-stranded DNA molecules internally labelled with two fixed fluorophores, we demonstrate the accuracy and precision of our method using the known structure of double-stranded DNA as a benchmark, resolve 10-base-pair differences in fluorophore separations, and determine the unique 3D orientation of each DNA molecule, thereby establishing short, double-labelled DNA molecules as probes of 3D orientation of anything to which one can attach them firmly.

Harmonic force spectroscopy measures load-dependent kinetics of individual human ß-cardiac myosin molecules.

Molecular motors are responsible for numerous cellular processes from cargo transport to heart contraction. Their interactions with other cellular components are often transient and exhibit kinetics that depend on load. Here, we measure such interactions using 'harmonic force spectroscopy'. In this method, harmonic oscillation of the sample stage of a laser trap immediately, automatically and randomly applies sinusoidally varying loads to a single motor molecule interacting with a single track along which it moves. The experimental protocol and the data analysis are simple, fast and efficient. The protocol accumulates statistics fast enough to deliver single-molecule results from single-molecule experiments. We demonstrate the method's performance by measuring the force-dependent kinetics of individual human ß-cardiac myosin molecules interacting with an actin filament at physiological ATP concentration. We show that a molecule's ADP release rate depends exponentially on the applied load, in qualitative agreement with cardiac muscle, which contracts with a velocity inversely proportional to external load.

Observation of correlated X-ray scattering at atomic resolution.

Tools to study disordered systems with local structural order, such as proteins in solution, remain limited. Such understanding is essential for e.g. rational drug design. Correlated X-ray scattering (CXS) has recently attracted new interest as a way to leverage next-generation light sources to study such disordered matter. The CXS experiment measures angular correlations of the intensity caused by the scattering of X-rays from an ensemble of identical particles, with disordered orientation and position. Averaging over 15 496 snapshot images obtained by exposing a sample of silver nanoparticles in solution to a micro-focused synchrotron radiation beam, we report on experimental efforts to obtain CXS signal from an ensemble in three dimensions. A correlation function was measured at wide angles corresponding to atomic resolution that matches theoretical predictions. These preliminary results suggest that other CXS experiments on disordered ensembles--such as proteins in solution--may be feasible in the future.

Single-molecule dual-beam optical trap analysis of protein structure and function.

Optical trapping is one of the most powerful single-molecule techniques. We provide a practical guide to set up and use an optical trap, applied to the molecular motor myosin as an example. We focus primarily on studies of myosin function using a dual-beam optical trap, a protocol to build such a trap, and the experimental and data analysis protocols to utilize it.