Single molecule physics and chemistry

New experiments using scanning probe microcopies and advanced optical methods allow us to study molecules as individuals, not just as populations. The findings of these studies not only include the confirmation of results expected from studies of bulk matter, but also give substantially new information concerning the complexity of biomolecules or molecules in a structured environment. The technique lays the groundwork for achieving the control of an individual molecule’s motion. Ultimately, this work may lead to such practical applications as miniaturized sensors.

An approach to three-dimensional structures of biomolecules by using single-molecule diffraction images

We describe an approach to the high-resolution three-dimensional structural determination of macromolecules that utilizes ultrashort, intense x-ray pulses to record diffraction data in combination with direct phase retrieval by the oversampling technique. It is shown that a simulated molecular diffraction pattern at 2.5-Å resolution accumulated from multiple copies of single rubisco biomolecules, each generated by a femtosecond-level x-ray free electron laser pulse, can be successfully phased and transformed into an accurate electron density map comparable to that obtained by more conventional methods. The phase problem is solved by using an iterative algorithm with a random phase set as an initial input. The convergence speed of the algorithm is reasonably fast, typically around a few hundred iterations. This approach and phasing method do not require any ab initio information about the molecule, do not require an extended ordered lattice array, and can tolerate high noise and some missing intensity data at the center of the diffraction pattern. With the prospects of the x-ray free electron lasers, this approach could provide a major new opportunity for the high-resolution three-dimensional structure determination of single biomolecules.

Single-molecule spectroscopy of the β2 adrenergic receptor: Observation of conformational substates in a membrane protein

Single-molecule studies of the conformations of the intact β2 adrenergic receptor were performed in solution. Photon bursts from the fluorescently tagged adrenergic receptor in a micelle were recorded. A photon-burst algorithm and a Poisson time filter were implemented to characterize single molecules diffusing across the probe volume of a confocal microscope. The effects of molecular diffusion and photon number fluctuations were deconvoluted by assuming that Poisson distributions characterize the molecular occupation and photon numbers. Photon-burst size histograms were constructed, from which the source intensity distributions were extracted. Different conformations of the β2 adrenergic receptor cause quenching of the bound fluorophore to different extents and hence produce different photon-burst sizes. An analysis of the photon-burst histograms shows that there are at least two distinct substates for the native adrenergic membrane receptor. This behavior is in contrast to one peak observed for the dye molecule, rhodamine 6G. We test the reliability and robustness of the substate number determination by investigating the application of different binning criteria. Conformational changes associated with agonist binding result in a marked change in the distribution of photon-burst sizes. These studies provide insight into the conformational heterogeneity of G protein-coupled receptors in the presence and absence of a bound agonist.

Imaging of single molecule diffusion.

In recent years observations at the level of individual atoms and molecules became possible by microscopy and spectroscopy. Imaging of single fluorescence molecules has been achieved but has so far been restricted to molecules in the immobile state. Here we provide methodology for visualization of the motion of individual fluorescent molecules. It is applied to imaging of the diffusional path of single molecules in a phospholipid membrane by using phospholipids carrying one rhodamine dye molecule. For this methodology, fluorescence microscopy was carried to a sensitivity so that single fluorescent molecules illuminated for only 5 ms were resolvable at a signal/noise ratio of 28. Repeated illuminations permitted direct observation of the diffusional motion of individual molecules with a positional accuracy of 30 nm. Such capability has fascinating potentials in bioscience--for example, to correlate biological functions of cell membranes with movements, spatial organization, and stoichiometries of individual components.

Stretching dependence of the vibration modes of a single-molecule Pt-H2-Pt bridge

A conducting bridge of a single hydrogen molecule between Pt electrodes is formed in a break junction experiment. It has a conductance near the quantum unit, G0=2e2∕h, carried by a single channel. Using point-contact spectroscopy three vibration modes are observed and their variation upon isotope substitution is obtained. The stretching dependence for each of the modes allows uniquely classifying them as longitudinal or transversal modes. The interpretation of the experiment in terms of a Pt-H2-Pt bridge is verified by density-functional theory calculations for the stability, vibrational modes, and conductance of the structure.

Mn-doped II-VI quantum dots: artificial molecular magnets

The notion of artificial atom relies on the capability to change the number of carriers one by one in semiconductor quantum dots, and the resulting changes in their electronic structure. Organic molecules with transition metal atoms that have a net magnetic moment and display hysteretic behaviour are known as single molecule magnets (SMM). The fabrication of CdTe quantum dots chemically doped with a controlled number of Mn atoms and with a number of carriers controlled either electrically or optically paves the way towards a new concept in nanomagnetism: the artificial single molecule magnet. Here we study the magnetic properties of a Mn-doped CdTe quantum dot for different charge states and show to what extent they behave like a single molecule magnet.

Single-molecule dynamic triangulation

A proposal for using single molecules as nanoprobes capable of detecting the trajectory of an elementary charge is discussed in detail. Presented numerical simulations prove that this singlemolecule technique allows determination of a three-dimensional single-electron displacement within a few seconds with an accurocy better than 0.006 nm. Surprisingly, this significantly exceeds the accuracy with which the probe;, molecule itself can be localized (given the same measuring time by means of single-molecule microscopy. It is also shown that the optimal concentration of probe molecules in the vicinity of:the electron (i.e. the concentration which provides the best accuracy of the inferred electron displacement) is of the order of 10(-5) m.

Kinetic modeling of molecular motors: pause model and parameter determination from single-molecule experiments

Single-molecule manipulation experiments of molecular motors provide essential information about the rate and conformational changes of the steps of the reaction located along the manipulation coordinate. This information is not always sufficient to define a particular kinetic cycle. Recent single-molecule experiments with optical tweezers showed that the DNA unwinding activity of a Phi29 DNA polymerase mutant presents a complex pause behavior, which includes short and long pauses. Here we show that different kinetic models, considering different connections between the active and the pause states, can explain the experimental pause behavior. Both the two independent pause model and the two connected pause model are able to describe the pause behavior of a mutated Phi29 DNA polymerase observed in an optical tweezers single-molecule experiment. For the two independent pause model all parameters are fixed by the observed data, while for the more general two connected pause model there is a range of values of the parameters compatible with the observed data (which can be expressed in terms of two of the rates and their force dependencies). This general model includes models with indirect entry and exit to the long-pause state, and also models with cycling in both directions. Additionally, assuming that detailed balance is verified, which forbids cycling, this reduces the ranges of the values of the parameters (which can then be expressed in terms of one rate and its force dependency). The resulting model interpolates between the independent pause model and the indirect entry and exit to the long-pause state model

The (Un)Folding of Multidomain Proteins Through the Lens of Single-molecule Force-spectroscopy and Computer Simulation

Proteins are specialized molecules that catalyze most of the reactions that can sustain life, and they become functional by folding into a specific 3D structure. Despite their importance, the question, "how do proteins fold?" - first pondered in in the 1930's - is still listed as one of the top unanswered scientific questions as of 2005, according to the journal Science. Answering this question would provide a foundation for understanding protein function and would enable improved drug targeting, efficient biofuel production, and stronger biomaterials. Much of what we currently know about protein folding comes from studies on small, single-domain proteins, which may be quite different from the folding of large, multidomain proteins that predominate the proteomes of all organisms.

In this thesis I will discuss my work to fill this gap in understanding by studying the unfolding and refolding of large, multidomain proteins using the powerful combination of single-molecule force-spectroscopy experiments and molecular dynamic simulations.

The three model proteins studied - Luciferase, Protein S, and Streptavidin - lend insight into the inter-domain dependence for unfolding and the subdomain stabilization of binding ligands, and ultimately provide new insight into atomistic details of the intermediate states along the folding pathway.

Single-molecule fluorescence resonance energy transfer study of SNARE-mediated membrane fusion

This is a comprehensive study of protein-mediated membrane fusion through single-molecule fluorescence resonance energy transfer (smFRET). Membrane fusion is one of the important cellular processes by which two initially distinct lipid bilayers merge their hydrophobic cores, resulting in one interconnected structure. For example, exocytosis, fertilization of an egg by a sperm and communication between neurons are a few among many processes that rely on some form of fusion. Proteins called soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) play a central role in fusion processes which is also regulated by many accessory proteins, such as synaptotagmin, complexin and Munc18. By a new lipid mixing method at the single-vesicle level, we are able to accurately detect different stages of SNARE-mediated membrane fusion including docking, hemi and full fusion via FRET value of single donor/acceptor vesicle pair. Through this single-vesicle lipid mixing assay, we discovered the vesicle aggregation induced by C2AB/Ca2+, the dual function of complexin, and the fusion promotion role of Munc18/SNARE-core binding mode. While this new method provides the information regarding the extent of the ensemble lipid mixing, the fusion pore opening between two vesicular cavities and the interaction between proteins cannot be detected. In order to overcome these limitations, we then developed a single-vesicle content mixing method to reveal the key factor of pore expansion by detecting the FRET change of dual-labeled DNA probes encapsulated in vesicles. Through our single-vesicle content mixing assay, we found the fusion pore expansion role of yeast SNAREs as well as neuronal SNAREs plus synaptotagmin 1.

Isotropic and anisotropic spin-spin interactions and a quantum phase transition in a dinuclear Cu(II) compound

We report electron-paramagnetic resonance (EPR) studies at similar to 9.5 GHz (X band) and similar to 34 GHz (Q band) of powder and single-crystal samples of the compound Cu(2)[TzTs](4) [N-thiazol-2-yl-toluenesulfonamidatecopper(II)], C(40)H(36)Cu(2)N(8)O(8)S(8), having copper(II) ions in dinuclear units. Our data allow determining an antiferromagnetic interaction J(0)=(-113 +/- 1) cm(-1) (H(ex)=-J(0)S(1)center dot S(2)) between Cu(II) ions in the dinuclear unit and the anisotropic contributions to the spin-spin coupling matrix D (H(ani)=S(1)center dot D center dot S(2)), a traceless symmetric matrix with principal values D/4=(0.198 +/- 0.003) cm(-1) and E/4=(0.001 +/- 0.003) cm(-1) arising from magnetic dipole-dipole and anisotropic exchange couplings within the units. In addition, the single-crystal EPR measurements allow detecting and estimating very weak exchange couplings between neighbor dinuclear units, with an estimated magnitude parallel to J(')parallel to=(0.060 +/- 0.015) cm(-1). The interactions between a dinuclear unit and the ""environment"" of similar units in the structure of the compound produce a spin dynamics that averages out the intradinuclear dipolar interactions. This coupling with the environment leads to decoherence, a quantum phase transition that collapses the dipolar interaction when the isotropic exchange coupling with neighbor dinuclear units equals the magnitude of the intradinuclear dipolar coupling. Our EPR experiments provide a new procedure to follow the classical exchange-narrowing process as a shift and collapse of the line structure (not only as a change of the resonance width), which is described with general (but otherwise simple) theories of magnetic resonance. Using complementary procedures, our EPR measurements in powder and single-crystal samples allow measuring simultaneously three types of interactions differing by more than three orders of magnitude (between 113 cm(-1) and 0.060 cm(-1)).

Ni(II)-doped CsCdBrCl2: Variation of spectral and structural properties via mixed-halide coordination

A new addition to the family of single-molecule magnets is reported: an Fete cage stabilized with benzoate and pyridonate ligands. Monte Carlo methods have been used to derive exchange parameters within the cage, and hence model susceptibility behavior.

Polynuclear Ni(II) Complexes with Schiff Bases as Bridging Ligands: A Molecular Approach to Nanoscience

The initial employment of N-salicylidene-2-amino-5-chlorobenzoic acid (sacbH2) as bridging/chelating ligand in metal cluster chemistry has provided access to five new polynuclear NiII complexes with large nuclearities, unprecedented metal core topologies, and interesting magnetic properties. The obtained results are presented in two projects. The first project includes the investigation of the general Ni2+/RCO2-/sacbH2 reaction system (where R- = CH3-, But-, ButCH2-) in which the nature of the carboxylic acid was found to be of crucial importance, affecting enormously the nuclearity of the resulting complexes. The second project deals with the study of the general Ni2+/X-/sacbH2 reaction system (where X- = inorganic anions) under basic conditions, yielding new cluster compounds with molecular chain-like structures and ferromagnetic exchange interactions between the metal centers.

Études de l’effet tunnel des spins quantiques macroscopiques

Dans cette thèse, nous présentons quelques analyses théoriques récentes ainsi que des observations expérimentales de l’effet tunnel quantique macroscopique et des tran- sitions de phase classique-quantique dans le taux d’échappement des systèmes de spins élevés. Nous considérons les systèmes de spin biaxial et ferromagnétiques. Grâce à l’approche de l’intégral de chemin utilisant les états cohérents de spin exprimés dans le système de coordonnées, nous calculons l’interférence des phases quantiques et leur distribution énergétique. Nous présentons une exposition claire de l’effet tunnel dans les systèmes antiferromagnétiques en présence d’un couplage d’échange dimère et d’une anisotropie le long de l’axe de magnétisation aisé. Nous obtenons l’énergie et la fonc- tion d’onde de l’état fondamentale ainsi que le premier état excité pour les systèmes de spins entiers et demi-entiers impairs. Nos résultats sont confirmés par un calcul utilisant la théorie des perturbations à grand ordre et avec la méthode de l’intégral de chemin qui est indépendant du système de coordonnées. Nous présentons aussi une explica- tion claire de la méthode du potentiel effectif, qui nous laisse faire une application d’un système de spin quantique vers un problème de mécanique quantique d’une particule. Nous utilisons cette méthode pour analyser nos modèles, mais avec la contrainte d’un champ magnétique externe ajouté. La méthode nous permet de considérer les transitions classiques-quantique dans le taux d’échappement dans ces systèmes. Nous obtenons le diagramme de phases ainsi que les températures critiques du passage entre les deux régimes. Nous étendons notre analyse à une chaine de spins d’Heisenberg antiferro- magnétique avec une anisotropie le long d’un axe pour N sites, prenant des conditions frontière périodiques. Pour N paire, nous montrons que l’état fondamental est non- dégénéré et donné par la superposition des deux états de Néel. Pour N impair, l’état de Néel contient un soliton, et, car la position du soliton est indéterminée, l’état fondamen- tal est N fois dégénéré. Dans la limite perturbative pour l’interaction d’Heisenberg, les fluctuations quantiques lèvent la dégénérescence et les N états se réorganisent dans une bande. Nous montrons qu’à l’ordre 2s, où s est la valeur de chaque spin dans la théorie des perturbations dégénérées, la bande est formée. L’état fondamental est dégénéré pour s entier, mais deux fois dégénéré pour s un demi-entier impair, comme prévu par le théorème de Kramer