No description available.

Single-molecule force spectroscopy (SMFS) is a unique approach to study the mechanical unfolding of proteins. SMFS unfolding experiments yield insight into how interactions stabilize a protein and guide its unfolding and refolding pathways. In contrast to various water-soluble proteins whose unfolding and refolding patterns have been characterized, only α-helical membrane proteins have been probed by SMFS. It was shown that α-helical membrane proteins unfold via many intermediates; this differs from the two-state unfolding process usually observed in water-soluble proteins. In membrane proteins, upon mechanically pulling the peptide end of the protein, single and grouped α-helices and polypeptide loops unfold in steps until the entire protein is unfolded. Whether the α-helices and loops unfold individually or cooperatively to form an unfolding intermediate depends on the interactions established within the membrane protein and the membrane. Each unfolding event relates to an unfolding intermediate with the sequence of these intermediates defining the unfolding pathway of the protein. β-barrel-forming membrane proteins are the second major group of membrane proteins and have not yet been studied by SMFS. To fill this void this study was designed to characterize interactions, unfolding, and refolding of the β-barrel forming outermembrane protein G (OmpG).Folding of transmembrane proteins, despite the important part these proteins play in every biological process in a cell, is studied in only a few examples. Of those only a handful were β-stranded membrane proteins (Tamm et al., 2004; Kleinschmidt et al., 2006). Current models describe that transmembrane β-barrels fold into the lipid membrane via two major steps. First the unfolded polypeptide interacts with the lipid surface where it then folds and inserts into the membrane (Kleinschmidt et al., 2006; Huysmans et al., 2010). Conventionally, thermal or chemical denaturation is used to study folding of membrane proteins. In most cases membrane proteins were solubilized in detergent or exposed to urea to be studied, conditions that are not compatible with In vivo conditions. This suggests that the folding pathways described so far may not be a realistic representation of such pathways in nature. SMFS represents a unique approach to study the unfolding and refolding of membrane proteins into the lipid membrane (Kedrov et al., 2006; Kessler et al., 2006). Using SMFS makes it possible to study unfolding and refolding of membrane proteins in their nativephysiological environment with controlled pH, electrolyte, temperature, and most importantly in the absence of any chemical denaturant or detergent. In this thesis, SMFS was utilized to unfold and refold OmpG in E coli lipid extract. Bulk unfolding experiments suggested that OmpG unfolds and folds reversibly and much faster than α-helical proteins (Conlan et al., 2000). The folding process is thought to be a coupled two-state membrane partition-folding reaction. To the contrary, the mechanical unfolding of OmpG consisted of many sequential unfolding intermediates. Our SMFS refolding experiments showed that a partially unfolded OmpG molecule also refolds via several sequential steps. The predominant refolding steps are defined by individual β-hairpins that could later assemble the transmembrane β-barrel of OmpG. In conclusion, the most probable unfolding and refolding pathways of OmpG as a membrane β-barrel protein go through the β-hairpins as the structural segments or unfolding-refolding intermediates and the process is a multi step one rather than the simple two state process. We also used SMFS to study the physical interactions that switch the functional state and gating of OmpG. The structural changes that gate OmpG have been previously described by X-ray crystallography (Yildiz et al., 2006). They showed when the pH changes from neutral to acidic the flexible extracellular loop L6 folds into the pore and closes the OmpG pore. Here, SMFS was used to structurally localize and quantify the interactions that are associated with the pH-dependent closure. At an acidic pH, a pH-dependent interaction at loop L6 was detected. This interaction changed the unfolding of loop L6 and β-strands 11 and 12, which connect loop L6. All other interactions detected within OmpG were found to be unaffected by changes in pH. These results provide a quantitative and mechanistic explanation of how pHdependent interactions change the folding of a peptide loop to gate the transmembrane pore. It has also been shown how the stability of OmpG is optimized so that pH changes modify only those interactions necessary to gate the transmembrane pore and there are no global changes in protein conformation or mechanical properties. In the next step of interactions study, dynamic SMFS (DFS) was applied to quantify the parameters characterizing the energy barriers in energy landscape for unfolding of the OmpG. Some of these parameters are: free energy of activation and distance of the transition state from the folded state. The pH-dependent functional switching of OmpG directs the protein along different regions at the unfolding energy landscape. The two functional states of OmpG sequential folding take the same unfolding pathway as β-hairpins I–IV. After the initial unfolding events, the unfolding pathways diverge. In the open state, the unfolding of β-hairpin V in one step precedes the unfolding of β-hairpin VI. In the closed state, β-hairpin V and β-strand S11 with a part of extracellular loop L6 unfold cooperatively, and subsequently β-strand S12 unfolds with the remaining loop L6. These two unfolding pathways in the open and closed states join again in the last unfolding step of β-hairpin VII. Also, the conformational change from the open to the closed state witnesses a difference in Xu and κ in the energy landscape that translates to rigidified extracellular loop L6 at the gating area. Thus, a change in the conformational state of OmpG not only bifurcates its unfolding pathways but also tunes its mechanical properties for optimum function.

The factored state representation and concurrency semantics of Petri nets are closely related to those of classical planning models, yet automated planning and Petri net analysis have developed independently, with minimal and mainly unconvincing attempts at crossfertilisation. This thesis exploits the relationship between the formal reachability problem, and the automated planning problem, via Petri net unfolding, which is an attractive reachability analysis method for highly concurrent systems as it facilitates reasoning about independent sub-problems. The first contribution of this thesis is the theory of directed unfolding: controlling the unfolding process with informative strategies, for the purpose of optimality and increased efficiency. The second contribution is the application of directed unfolding to automated planning. Inspired by well-known planning heuristics, this thesis shows how problem specific information can be employed to guide unfolding, in response to the formal problem of developing efficient, directed reachability analysis methods for concurrent systems. Complimenting this theoretical work, this thesis presents a new forward search method for partial order planning which can be exponentially more efficient than state space search. Our suite of planners based on directed unfolding can perform optimal and suboptimal classical planning subject to arbitrary action costs, optimal temporal planning with respect to arbitrary action durations, and address probabilistic planning via replanning for the most likely path. Empirical results reveal directed unfolding is competitive with current state of the art automated planning systems, and can solve Petri net reachability problems beyond the reach of the original “blind” unfolding technique. / Thesis (Ph.D.) -- University of Adelaide, School of Electrical and Electronic Engineering, 2008

Le rayonnement cosmique, mis en évidence par Viktor Hess en 1912, est composé de particules chargées, créées et possiblement accélérées dans les restes de supernova, et qui se propagent dans la Galaxie. La mesure des flux du rayonnement cosmique permet de mettre des contraintes sur leurs sources et leur transport, mais aussi de se pencher sur le problème de la matière sombre.C'est pour répondre à ces questions qu'a été construit le détecteur AMS-02, mis en place sur la station spatiale internationale depuis mai 2011. Ce détecteur de haute précision mesure l'ensemble des flux de particules du rayonnement cosmique.Le travail proposé dans cette thèse consiste à estimer le flux de protons avec le détecteur AMS-02. L'accent est mis sur la déconvolution des effets de la réponse en énergie du détecteur sur les flux et sur la caractérisation du flux obtenu à haute énergie (au-dessus de 200 GeV/n) avec la mise en évidence d'une cassure spectrale.D'autre part, le soleil émet un plasma qui interagit avec les particules du rayonnement cosmique, modifiant les flux issus de la propagation dans la Galaxie. Cette modification évolue dans le temps en suivant le cycle d'activité solaire et est appelée modulation solaire. Dans ce cadre, nous avons obtenu une nouvelle détermination robuste des flux interstellaires de protons et d'hélium en nous basant sur les données récentes du rayonnement cosmique (incluant AMS-02). Les niveaux de modulation solaire obtenus sont validés avec une seconde analyse réalisée à partir des données des moniteurs à neutrons, détecteurs au sol, qui permettent d'établir des séries en temps du paramètre de modulation depuis les années 50. / Cosmic rays (CR) were discovered by Viktor Hess in 1912. Charged CR are synthesized and supposedly accelerated in supernova remnants, then propagate through the Galaxy. CR flux measurement set constraints on CR sources and propagation, but may also bring answers to the dark matter problem.AMS-02 is a high precision particle physics detector placed on the international space station since may 2011. It measures the CR fluxes of many species.This thesis deals with the proton flux estimation measured by the AMS-02 instrument. The focus is set on the unfolding of the instrument energy response impacting the flux, and on the caracterisation of the high-energy spectral break.The Sun produces a plasma which interacts with CR particles, modifying the flux obtained from galactic propagation. This modification evolves through time following the solar activity cycle, and is denoted solar modulation. In this framework, decolving from this effect, a robust determination of the proton and helium interstellar fluxes is obtained using recent high precision CR data including AMS-02. The associated solar modulation levels are cross-checked with a second estimation taken from neutron monitors (ground based detectors) data, allowing solar modulation time series reconstruction from the 50s.

Made available in DSpace on 2014-12-17T14:06:51Z (GMT). No. of bitstreams: 1 AntonioEFF.pdf: 720128 bytes, checksum: febce55b295b3d3be15f1c24bf498884 (MD5) Previous issue date: 2009-02-26 / Na unfolding method of linear intercept distributions and secction ?rea distribution was implemented for structures with spherical grains. Although the unfolding routine depends on the grain shape, structures with spheroidal grains can also be treated by this routine. Grains of non-spheroidal shape can be treated only as approximation. A software was developed with two parts. The first part calculates the probability matrix. The second part uses this matrix and minimizes the chi-square. The results are presented with any number of size classes as required. The probability matrix was determined by means of the linear intercept and section area distributions created by computer simulation. Using curve fittings the probability matrix for spheres of any sizes could be determined. Two kinds of tests were carried out to prove the efficiency of the Technique. The theoretical tests represent ideal cases. The software was able to exactly find the proposed grain size distribution. In the second test, a structure was simulated in computer and images of its slices were used to produce the corresponding linear intercept the section area distributions. These distributions were then unfolded. This test simulates better reality. The results show deviations from the real size distribution. This deviations are caused by statistic fluctuation. The unfolding of the linear intercept distribution works perfectly, but the unfolding of section area distribution does not work due to a failure in the chi-square minimization. The minimization method uses a matrix inversion routine. The matrix generated by this procedure cannot be inverted. Other minimization method must be used / A rotina de desdobramento (unfolding) de distribui??es de intercepto linear e de ?rea de se??o foi implementada para gr?os de formato esf?rico. Apesar da rotina de unfolding ser fortemente dependente do formato do gr?o, estruturas que possuam gr?os com formatos esferoidais podem ser tratadas com esta rotina. Mesmo estruturas com gr?os de formatos n?o esferoidais podem ser tratadas por aproxima??o. Um programa com duas partes foi desenvolvido. Primeira parte determina a tabela de probabilidades. A segunda utiliza esta tabela e aplica o m?todo de minimiza??o do chi-quadrado. Os resultados s?o dados em qualquer n?mero de classes de tamanho de gr?o requerido pelo usu?rio. A tabela de probabilidade foi determinada a partir de distribui??es de intercepto linear e de ?rea de se??o geradas por simula??o computacional. Por meio de ajustes de curvas de distribui??o, tabelas de probabilidades para esferas de qualquer tamanho podem ser determinadas. Dois tipos de testes foram executados para verificar a efici?ncia do m?todo. Os testes te?ricos representam situa??es ideais. O programa conseguiu reproduzir com exatid?o as distribui??es de tamanho de gr?o sugeridas. Os testes simulados consistem em simular em computador distribui??es de tamanho de gr?os e executar todo o procedimento metalogr?fico usual. Este tipo de teste ? mais pr?ximo da situa??o real. Os resultados deste tipo de teste mostram que a rotina de medi??o estereol?gica introduz desvios estat?sticos, afastando o resultado encontrado do valor real. Contudo, a rotina de unfolding funciona perfeitamente para a distribui??o de intercepto linear. No caso de unfolding de ?rea de se??o, a minimiza??o do chi-quadrado pelo m?todo matricial gera matrizes n?o invers?veis e n?o pode ser aplicada. Outro m?todo de minimiza??o deve ser buscado

Single-molecule force spectroscopy (SMFS) is a unique approach to study the mechanical unfolding of proteins. SMFS unfolding experiments yield insight into how interactions stabilize a protein and guide its unfolding and refolding pathways. In contrast to various water-soluble proteins whose unfolding and refolding patterns have been characterized, only α-helical membrane proteins have been probed by SMFS. It was shown that α-helical membrane proteins unfold via many intermediates; this differs from the two-state unfolding process usually observed in water-soluble proteins. In membrane proteins, upon mechanically pulling the peptide end of the protein, single and grouped α-helices and polypeptide loops unfold in steps until the entire protein is unfolded. Whether the α-helices and loops unfold individually or cooperatively to form an unfolding intermediate depends on the interactions established within the membrane protein and the membrane. Each unfolding event relates to an unfolding intermediate with the sequence of these intermediates defining the unfolding pathway of the protein. β-barrel-forming membrane proteins are the second major group of membrane proteins and have not yet been studied by SMFS. To fill this void this study was designed to characterize interactions, unfolding, and refolding of the β-barrel forming outermembrane protein G (OmpG).Folding of transmembrane proteins, despite the important part these proteins play in every biological process in a cell, is studied in only a few examples. Of those only a handful were β-stranded membrane proteins (Tamm et al., 2004; Kleinschmidt et al., 2006). Current models describe that transmembrane β-barrels fold into the lipid membrane via two major steps. First the unfolded polypeptide interacts with the lipid surface where it then folds and inserts into the membrane (Kleinschmidt et al., 2006; Huysmans et al., 2010). Conventionally, thermal or chemical denaturation is used to study folding of membrane proteins. In most cases membrane proteins were solubilized in detergent or exposed to urea to be studied, conditions that are not compatible with In vivo conditions. This suggests that the folding pathways described so far may not be a realistic representation of such pathways in nature. SMFS represents a unique approach to study the unfolding and refolding of membrane proteins into the lipid membrane (Kedrov et al., 2006; Kessler et al., 2006). Using SMFS makes it possible to study unfolding and refolding of membrane proteins in their nativephysiological environment with controlled pH, electrolyte, temperature, and most importantly in the absence of any chemical denaturant or detergent. In this thesis, SMFS was utilized to unfold and refold OmpG in E coli lipid extract. Bulk unfolding experiments suggested that OmpG unfolds and folds reversibly and much faster than α-helical proteins (Conlan et al., 2000). The folding process is thought to be a coupled two-state membrane partition-folding reaction. To the contrary, the mechanical unfolding of OmpG consisted of many sequential unfolding intermediates. Our SMFS refolding experiments showed that a partially unfolded OmpG molecule also refolds via several sequential steps. The predominant refolding steps are defined by individual β-hairpins that could later assemble the transmembrane β-barrel of OmpG. In conclusion, the most probable unfolding and refolding pathways of OmpG as a membrane β-barrel protein go through the β-hairpins as the structural segments or unfolding-refolding intermediates and the process is a multi step one rather than the simple two state process. We also used SMFS to study the physical interactions that switch the functional state and gating of OmpG. The structural changes that gate OmpG have been previously described by X-ray crystallography (Yildiz et al., 2006). They showed when the pH changes from neutral to acidic the flexible extracellular loop L6 folds into the pore and closes the OmpG pore. Here, SMFS was used to structurally localize and quantify the interactions that are associated with the pH-dependent closure. At an acidic pH, a pH-dependent interaction at loop L6 was detected. This interaction changed the unfolding of loop L6 and β-strands 11 and 12, which connect loop L6. All other interactions detected within OmpG were found to be unaffected by changes in pH. These results provide a quantitative and mechanistic explanation of how pHdependent interactions change the folding of a peptide loop to gate the transmembrane pore. It has also been shown how the stability of OmpG is optimized so that pH changes modify only those interactions necessary to gate the transmembrane pore and there are no global changes in protein conformation or mechanical properties. In the next step of interactions study, dynamic SMFS (DFS) was applied to quantify the parameters characterizing the energy barriers in energy landscape for unfolding of the OmpG. Some of these parameters are: free energy of activation and distance of the transition state from the folded state. The pH-dependent functional switching of OmpG directs the protein along different regions at the unfolding energy landscape. The two functional states of OmpG sequential folding take the same unfolding pathway as β-hairpins I–IV. After the initial unfolding events, the unfolding pathways diverge. In the open state, the unfolding of β-hairpin V in one step precedes the unfolding of β-hairpin VI. In the closed state, β-hairpin V and β-strand S11 with a part of extracellular loop L6 unfold cooperatively, and subsequently β-strand S12 unfolds with the remaining loop L6. These two unfolding pathways in the open and closed states join again in the last unfolding step of β-hairpin VII. Also, the conformational change from the open to the closed state witnesses a difference in Xu and κ in the energy landscape that translates to rigidified extracellular loop L6 at the gating area. Thus, a change in the conformational state of OmpG not only bifurcates its unfolding pathways but also tunes its mechanical properties for optimum function.:Table of Contents INTRODUCTION:1 1.1 THE FIRST UNIT OF LIFE STARTED WITH MEMBRANE:1 1.2.1 CELL MEMBRANE STRUCTURE: 2 1.3 MEMBRANE PROTEINS:3 1.3.1 α-­‐HELICAL MEMBRANE PROTEINS:5 1.3.2 β-­‐BARREL MEMBRANE PROTEIN:5 1.4 MEMBRANE PROTEINS FOLDING:12 1.4.1 MODELS FOR α-­‐HELICAL MEMBRANE PROTEIN FOLDING:13 1.4.2 MODELS FOR β-­‐BARREL MEMBRANE PROTEIN FOLDING:15 1.5. GATING STUDY OF MEMBRANE PROTEINS:18 ATOMIC FORCE MICROSCOPY:19 2.1 ATOMIC FORCE MICROSCOPE:19 2.1.1 HISTORY:19 2.1.2 PRINCIPLE:19 2.1.3 THE CANTILEVER:20 2.1.4 AFM MODES 23 2.2 SINGLE-­‐MOLECULE FORCE SPECTROSCOPY:25 2.2.1 DYNAMIC FORCE SPECTROSCOPY,(DYNAMIC SMFS):27 2.3 WHAT IS THE ADVANTAGE OF USING ATOMIC FORCE MICROSCOPY IN MEMBRANE PROTEIN STUDIES?:29 FOLDING MECHANISM OF OMPG:31 3.1 UNFOLDING PATTERN: ONEβ-­‐HAIRPIN AFTER THE OTHER:31 3.1.1 OUTER MEMBRANE PROTEIN G (OMPG):31 3.1.2 MECHANICAL UNFOLDING PATHWAYS OF THE MEMBRANE β-­‐BARREL PROTEIN OMPG:33 3.1.3 MATERIAL AND METHODS:34 3.1.4 RESULTS AND DISCUSSION:41 3.2 REFOLDING PATTERN: ONE Β-­‐HAIRPIN AFTER THE OTHER:48 3.2.1. EXPLORING REFOLDING PATHWAYS AND KINETICS OF THE MEMBRANE Β-­‐BARREL PROTEIN OMPG:48 3.2.2 EXPERIMENTAL PROCEDURES:49 3.2.3 RESULTS:50 3.2.4 DISCUSSION:52 INTERACTION STUDIES:59 4.1 PH-­‐DEPENDENT INTERACTIONS GUIDE THE FOLDING AND GATE THE TRANSMEMBRANE PORE OF THE β-­‐BARREL TRANSMEMBRANE PROTEIN OMPG:59 4.1.2 INTRODUCTION:59 4.1.2 EXPERIMENTAL PROCEDURES:61 4.1.3 RESULTS AND DISCUSSION:62 4.2 DUAL ENERGY LANDSCAPE: THE FUNCTIONAL STATE OF THE OUTER MEMBRANE β-­‐BARREL PROTEIN OMPG MOLDS ITS UNFOLDING ENERGY LANDSCAPE:67 4.2.1 INTRODUCTION:67 4.2.2 EXPERIMENTAL PROCEDURES:71 4.2.3 RESULTS AND DISCUSSION:74 4.2.3.1 FUNCTIONAL STATE OF OMPG DIRECTS ITS UNFOLDING ROUTE:74 4.2.3.2 QUANTIFYING THE UNFOLDING ENERGY BARRIERS OF OMPG IN THE CLOSED AND OPEN CONFORMATIONS:75 4.2.3.3 TRANSITION STATE DISTANCES OF UNFOLDING ENERGY BARRIERS:77 4.2.3.4 ACTIVATION FREE ENERGY OF Β-­‐STRANDS AND Β-­‐HAIRPINS:79 4.2.3.5 MECHANICAL PROPERTIES OF OMPG:83 4.2.3.6 MAPPING THE UNFOLDING ENERGY LANDSCAPES OF OMPG IN THE OPEN AND CLOSED STATES:85 4.2.4 CONCLUSION:86 OUTLOOK:89 5.1 INTRODUCTION:89 5.2 INTERACTION STUDY AND UNFOLDING ENERGY LANDSCAPE:90 5.3 MEMBRANE PROTEINF OLDING:92 REFRENCES:96 ABBREVIATIONS:110 SYMBOLS:111 PUBLICATIONS:113 ACKNOWLEDGMENT:114 DECLARATION: 115

L’expérience Tokai-to-Kamioka (T2K), située au Japon, étudie les oscillations des neutrinos et anti-neutrinos muoniques. Elle se donne pour objectif de mesurer les paramètres de la matrice de mélange, en particulier l’angle de mélange θ13 et la phase de violation de la symétrie CP. L’incertitude principale sur ces mesures provient de la méconnaissance des interactions des neutrinos avec les noyaux des divers matériaux composant les détecteurs. Afin de réduire ces erreurs systématiques, le détecteur WAGASCI, composé d’un réseau de barres de scintillateurs dans une cuve d’eau, a été construit et installé en 2016 sur le site de J-PARC à Tokai.Nous présentons dans cette thèse le phénomène d’oscillation des neutrinos et les effets nucléaires qui permettent de décrire les interactions entre neutrinos et noyaux. Nous illustrons également la construction du premier module WAGASCI – appelé le WaterModule – effectuée à l’automne 2015 ainsi que les études d’étalonnage de la réponse en énergie du détecteur.Les données collectées avec le WaterModule sont ensuite utilisées pour mesurer la section efficace des interactions par courant chargé des neutrinos muoniques produisant un pion dans l’état final (canal CC1π). Nous présentons ainsi les étapes menant à cette mesure : l’identification des particules, la sélection d’un échantillon d’évènements candidats puis l’étude des incertitudes statistiques et systématiques. Pour extraire la section efficace en fonction de l’angle et de l’impulsion du muon, nous utilisons une méthode statistique (unfolding) itérative pour laquelle un critère de convergence doit être établi ; nous présentons cette méthode ainsi qu’un moyen de construire un critère de convergence dicté par les données.Cette mesure, comme l’ensemble des mesures effectuées avec le détecteur WAGASCI, contribuera à réduire de manière significative les incertitudes systématiques de l’expérience T2K, ouvrant la voie à la mesure de la phase de violation de la symétrie CP. / The Tokai-to-Kamioka experiment (T2K), located in Japan, studies oscillations of muon neutrinos and antineutrinos. It aims to measure neutrino mixing parameters, such as the mixing angle θ13 and the CP-symmetry violating phase. The principal uncertainty on these measurements relates to the limited knowledge on neutrino-nucleus interactions on various target materials in the detectors. In order to reduce these systematic uncertainties, the WAGASCI detector – a lattice of scintillator bars in a water tank – has been built and installed in 2016 at J-PARC (Tokai, Japan).In this thesis we introduce neutrino oscillations alongside the nuclear effects required to describe how neutrinos interact with nuclei. We also show how the first WAGASCI module, the so-called WaterModule, was built in autumn 2015 in Tokai. The charge calibration of the WaterModule is also presented.The first WaterModule data are used to measure the muon neutrino charged-current cross section with one charged pion in the final state (CC1π channel). We detail the steps leading to this measurement: the particle identification; the selection of CC1π-candidate events; and the study of statistical and systematic uncertainties. To extract the double differential cross section with respect to muon momentum and angle, we use an iterative unfolding approach that requires a convergence criterion. We present this method and a way to build a data-driven convergence criterion.This measurement, as well as all coming measurements from the full WAGASCI detector, will contribute to significantly reduce the systematic errors for the T2K experiment. We will thus take a step further towards the measurement of the CP-symmetry violating phase.

No description available.

Doctor of Philosophy / Department of Chemical Engineering / Peter H. Pfromm / One area of considerable importance in modern biotechnology is the preparation of highly active and selective enzyme based biocatalysts for applications in organic solvents. A major challenge is posed by the tendency of enzymes to cluster when suspended in organic solvents. Because the clusters obstruct the transport of substrates to the active site of the enzyme, the observed activity is often severely reduced. Over the past two decades, many strategies have been proposed to mitigate this problem. We have tackled this major hurdle by devising an immobilization strategy that utilizes fumed silica as carrier for the enzyme molecules. Fumed silica is a non-porous nanoparticulated fractal aggregate with unique absorptive properties. The enzyme/fumed silica preparation is formed in two steps. The buffered enzyme molecules are physically adsorbed on the fumed silica and then lyophilized. This protocol was shown to be successful with two enzymes of industrial relevance, Candida antarctica Lipase B (CALB) and subtilisin Carlsberg. The maximum observed catalytic activity in hexane reached or even exceeded commercial immobilizates and nonbuffer salt based preparations. The results demonstrated that catalytic activity has an intricate relationship with the nominal surface coverage (%SC) of the support by the enzyme molecules. s. Carlsberg exhibited an ever increasing activity as more surface area was provided per enzyme molecule. The activity leveled off when a sparse surface population was reached. CALB showed a maximum in catalytic activity at an intermediate surface coverage with steep decreases at both lower and higher surface coverage. It was shown that this maximum results from the presence of three distinct surface loading regimes after lyophilization: 1. a low surface coverage where opportunities for multi-attachment to the surface likely lead to detrimental conformational changes, 2. an intermediate surface coverage where interactions with neighboring proteins and the surface help to maintain a higher population of catalytically competent enzyme molecules, and 3. a multi-layer coverage where mass transfer limitations lead to a decrease in the apparent catalytic activity. Conformational stability analyses with both fluorescence and CD spectroscopy showed evidence that these regimes are most likely formed during the adsorption step of our protocol. A low conformational stability region was detected at low surface coverage while adsorbates with highly stable enzyme ensembles were observed at high surface coverage. Secondary structural analysis of the lyophilized nanobiocatalysts with FTIR confirmed a substantial decrease in the alpha-helical components at low surface coverage. In summary, the work presented here traces the phenomenological observation of the catalytic behavior of a nanobiocatalyst to molecular-level: enzyme-enzyme and enzyme-support interactions, which are specific to the intricate properties of the enzyme molecules.

The Gram-positive pathogens Corynebacterium diphtheriae and Streptococcus pyogenes both require iron for survival. These bacteria have developed sophisticated heme uptake and transport protein machinery responsible for the import of iron into the cell, in the form of heme from the human host. The heme utilization pathway (hmu) of C. diphtheriae utilizes multiple proteins to bind and transport heme into the cell. One of these proteins, HmuT, delivers heme to the ABC transporter HmuUV. The axial ligation of the heme in HmuT was probed by examination of wild-type HmuT and a series of conserved heme pocket residue mutants, H136A, Y235A, R237A, Y272A, M292A, Y349A, and Y349F. Characterization by UV-visible absorption, resonance Raman, and magnetic circular dichroism spectroscopies indicated that H136 and Y235 are the axial ligands in HmuT. Electrospray ionization mass spectrometry was also utilized to assess the roles of conserved residues in contribution to heme binding. The S. pyogenes streptococcal iron acquisition (sia)/heme transport system (hts) utilizes multiple proteins to bring host heme to the intracellular space. Both the substrate binding protein SiaA and the hemoprotein surface receptor Shr were investigated. The kinetic effects on SiaA heme release were probed through chemical unfolding of axial ligand mutants M79A and H229A, as well mutants thought to contribute to heme binding, K61A and C58A, and a control mutant, C47A. The unfolding pathways showed two processes for protein denaturation. This is consistent with heme loss from protein forms differing by the orientation of the heme in the binding pocket. The ease of protein unfolding is related to the strength of interaction of the residues with the heme. Shr contains two NEAT (near-iron transporter) domains (Shr-N1 and Shr-N2) which can both bind heme. Biophysical studies of both Shr-N1 and Shr-N2 indicated a new class of NEAT domains which utilize methionine as an axial ligand, rather than a tyrosine. Thermal and chemical unfolding showed ferrous Shr-N1 and Shr-N2 to be most resistant to denaturation. Shr-N2 was prone to autoreduction. Together, sequence alignment, homology modeling, and spectral signatures are all consistent with two methionines as the heme ligands of this novel type of NEAT heme-binding domain.