Electrochemical single-molecule transistors with optimized gate coupling

Electrochemical gating at the single molecule level of viologen molecular bridges in ionic liquids is examined. Contrary to previous data recorded in aqueous electrolytes, a clear and sharp peak in the single molecule conductance versus electrochemical potential data is obtained in ionic liquids. These data are rationalized in terms of a two-step electrochemical model for charge transport across the redox bridge. In this model the gate coupling in the ionic liquid is found to be fully effective with a modeled gate coupling parameter, ξ, of unity. This compares to a much lower gate coupling parameter of 0.2 for the equivalent aqueous gating system. This study shows that ionic liquids are far more effective media for gating the conductance of single molecules than either solid-state three-terminal platforms created using nanolithography, or aqueous media.

Automated NMR structure determination and disulfide bond identification of the myotoxin crotamine from Crotalus durissus terrificus

Crotamine is one of four major components of the venom of the South American rattlesnake Crotalus durissus terrificus. Similar to its counterparts in the family of the myotoxins, it induces myonecrosis of skeletal muscle cells. This paper describes a new NMR structure determination of crotamine in aqueous solution at pH 5.8 and 20 degrees C, using standard homonuclear (1)H NMR spectroscopy at 900 MHz and the automated structure calculation software ATNOS/CANDID/DYANA. The automatic NOESY spectral analysis included the identification of a most likely combination of the six cysteines into three disulfide bonds, i.e. Cys4-Cys36, Cys11-Cys30 and Cys18-Cys37; thereby a generally applicable new computational protocol is introduced to determine unknown disulfide bond connectivities in globular proteins. A previous NMR structure determination was thus confirmed and the structure refined. Crotamine contains an alpha-helix with residues 1-7 and a two-stranded anti-parallel beta-sheet with residues 9-13 and 34-38 as the only regular secondary structures. These are connected with each other and the remainder of the polypeptide chain by the three disulfide bonds, which also form part of a central hydrophobic core. A single conformation was observed, with Pro13 and Pro21 in the trans and Pro20 in the cis-form. The global fold and the cysteine-pairing pattern of crotamine are similar to the beta-defensin fold, although the two proteins have low sequence homology, and display different biological activities. (c) 2005 Elsevier Ltd. All rights reserved.

Assignment of the disulfide bridges in bothropstoxin-I, a myonecrotic Lys49 PLA(2) homolog from Bothrops jararacussu snake venom

Bothropstoxin-I (BthTX-1), a Lys49 phospholipase A(2) homolog with no apparent catalytic activity, was first isolated from Bothrops jararacussu snake venom and completely sequenced in this laboratory. It is a 121-amino-acid single polypeptide chain, highly myonecrotic, despite its inability to catalyze hydrolysis of egg yolk phospholipids, and has 14 half-cystine residues identified at positions 27, 29, 44, 45, 50, 51, 61, 84, 91, 96, 98, 105, 123, and 131 (numbering according to the conventional alignment including gaps, so that the last residue is Cys 131). In order to access its seven disulfide bridges, two strategies were followed: (1) Sequencing of isolated peptides from (tryptic + SV8) and chymotryptic digests by Edman-dansyl degradation; (2) crystallization of the protein and determination of the crystal structure so that at least two additional disulfide bridges could be identified in the final electron density map. Identification of the disulfide-containing peptides from the enzymatic digests was achieved following the disappearance of the original peptides from the HPLC profile after reduction and carboxymethylation of the digest. Following this procedure, four bridges were initially identified from the tryptic and SV8 digests: Cys50-Cys131, Cys51-Cys98, Cys61-Cys91, and Cys84-Cys96. From the chymotryptic digest other peptides were isolated either containing some of the above bridges, therefore confirming the results from the tryptic digest, or presenting a new bond between Cys27 and Cys123. The two remaining bridges were identified as Cys29-Cys45 and Cys44-Cys105 by determination of the crystal structure, showing that BthTX-1 disulfide bonds follow the normal pattern of group II PLA(2)s.

Structure of a novel class II phospholipase D: Catalytic cleft is modified by a disulphide bridge

Phospholipases D (PLDs) are principally responsible for the local and systemic effects of Loxosceles envenomation including dermonecrosis and hemolysis. Despite their clinical relevance in loxoscelism, to date, only the SMase I from Loxosceles laeta, a class I member, has been structurally characterized. The crystal structure of a class II member from Loxosceles intermedia venom has been determined at 1.7. Å resolution. Structural comparison to the class I member showed that the presence of an additional disulphide bridge which links the catalytic loop to the flexible loop significantly changes the volume and shape of the catalytic cleft. An examination of the crystal structures of PLD homologues in the presence of low molecular weight compounds at their active sites suggests the existence of a ligand-dependent rotamer conformation of the highly conserved residue Trp230 (equivalent to Trp192 in the glycerophosphodiester phosphodiesterase from Thermus thermophofilus, PDB code: 1VD6) indicating its role in substrate binding in both enzymes. Sequence and structural analyses suggest that the reduced sphingomyelinase activity observed in some class IIb PLDs is probably due to point mutations which lead to a different substrate preference. © 2011 Elsevier Inc.

Each one teaches one: characterizing active forms of proteins by single molecule force spectroscopy

This Ph.D. candidate thesis collects the research work I conducted under the supervision of Prof.Bruno Samor´ı in 2005,2006 and 2007. Some parts of this work included in the Part III have been begun by myself during my undergraduate thesis in the same laboratory and then completed during the initial part of my Ph.D. thesis: the whole results have been included for the sake of understanding and completeness. During my graduate studies I worked on two very different protein systems. The theorical trait d’union between these studies, at the biological level, is the acknowledgement that protein biophysical and structural studies must, in many cases, take into account the dynamical states of protein conformational equilibria and of local physico-chemical conditions where the system studied actually performs its function. This is introducted in the introductory part in Chapter 2. Two different examples of this are presented: the structural significance deriving from the action of mechanical forces in vivo (Chapter 3) and the complexity of conformational equilibria in intrinsically unstructured proteins and amyloid formation (Chapter 4). My experimental work investigated both these examples by using in both cases the single molecule force spectroscopy technique (described in Chapter 5 and Chapter 6). The work conducted on angiostatin focused on the characterization of the relationships between the mechanochemical properties and the mechanism of action of the angiostatin protein, and most importantly their intertwining with the further layer of complexity due to disulfide redox equilibria (Part III). These studies were accompanied concurrently by the elaboration of a theorical model for a novel signalling pathway that may be relevant in the extracellular space, detailed in Chapter 7.2. The work conducted on -synuclein (Part IV) instead brought a whole new twist to the single molecule force spectroscopy methodology, applying it as a structural technique to elucidate the conformational equilibria present in intrinsically unstructured proteins. These equilibria are of utmost interest from a biophysical point of view, but most importantly because of their direct relationship with amyloid aggregation and, consequently, the aetiology of relevant pathologies like Parkinson’s disease. The work characterized, for the first time, conformational equilibria in an intrinsically unstructured protein at the single molecule level and, again for the first time, identified a monomeric folded conformation that is correlated with conditions leading to -synuclein and, ultimately, Parkinson’s disease. Also, during the research work, I found myself in the need of a generalpurpose data analysis application for single molecule force spectroscopy data analysis that could solve some common logistic and data analysis problems that are common in this technique. I developed an application that addresses some of these problems, herein presented (Part V), and that aims to be publicly released soon.

Comparison between analytical and numerical methods for the assessment of masonry arch bridges: Case study of Clemente Bridge on Savio river

This research has focused on the study of the behavior and of the collapse of masonry arch bridges. The latest decades have seen an increasing interest in this structural type, that is still present and in use, despite the passage of time and the variation of the transport means. Several strategies have been developed during the time to simulate the response of this type of structures, although even today there is no generally accepted standard one for assessment of masonry arch bridges. The aim of this thesis is to compare the principal analytical and numerical methods existing in literature on case studies, trying to highlight values and weaknesses. The methods taken in exam are mainly three: i) the Thrust Line Analysis Method; ii) the Mechanism Method; iii) the Finite Element Methods. The Thrust Line Analysis Method and the Mechanism Method are analytical methods and derived from two of the fundamental theorems of the Plastic Analysis, while the Finite Element Method is a numerical method, that uses different strategies of discretization to analyze the structure. Every method is applied to the case study through computer-based representations, that allow a friendly-use application of the principles explained. A particular closed-form approach based on an elasto-plastic material model and developed by some Belgian researchers is also studied. To compare the three methods, two different case study have been analyzed: i) a generic masonry arch bridge with a single span; ii) a real masonry arch bridge, the Clemente Bridge, built on Savio River in Cesena. In the analyses performed, all the models are two-dimensional in order to have results comparable between the different methods taken in exam. The different methods have been compared with each other in terms of collapse load and of hinge positions.

The effect of osmolytes on protein fold stability at the single-molecule level

By pulling and releasing the tension on protein homomers with the Atomic Force Miscroscope (AFM) at different pulling speeds, dwell times and dwell distances, the observed force-response of the protein can be fitted with suitable theoretical models. In this respect we developed mathematical procedures and open-source computer codes for driving such experiments and fitting Bell’s model to experimental protein unfolding forces and protein folding frequencies. We applied the above techniques to the study of proteins GB1 (the B1 IgG-binding domain of protein G from Streptococcus) and I27 (a module of human cardiac titin) in aqueous solutions of protecting osmolytes such as dimethyl sulfoxide (DMSO), glycerol and trimethylamine N-oxide (TMAO). In order to get a molecular understanding of the experimental results we developed an Ising-like model for proteins that incorporates the osmophobic nature of their backbone. The model benefits from analytical thermodynamics and kinetics amenable to Monte-Carlo simulation. The prevailing view used to be that small protecting osmolytes bridge the separating beta-strands of proteins with mechanical resistance, presumably shifting the transition state to significantly higher distances that correlate with the molecular size of the osmolyte molecules. Our experiments showed instead that protecting osmolytes slow down protein unfolding and speed-up protein folding at physiological pH without shifting the protein transition state on the mechanical reaction coordinate. Together with the theoretical results of the Ising-model, our results lend support to the osmophobic theory according to which osmolyte stabilisation is a result of the preferential exclusion of the osmolyte molecules from the protein backbone. The results obtained during this thesis work have markedly improved our understanding of the strategy selected by Nature to strengthen protein stability in hostile environments, shifting the focus from hypothetical protein-osmolyte interactions to the more general mechanism based on the osmophobicity of the protein backbone.

Balloon Aortic Valvuloplasty as Bridge-to-Decision in High Risk Patients with Severe Aortic Valve Stenosis

Background. A sizable group of patients with symptomatic aortic stenosis (AS) can undergo neither surgical aortic valve replacement (AVR) nor transcatheter aortic valve implantation (TAVI) because of clinical contraindications. The aim of this study was to assess the potential role of balloon aortic valvuloplasty (BAV) as a “bridge-to-decision” in selected patients with severe AS and potentially reversible contraindications to definitive treatment. Methods. We retrospectively enrolled 645 patients who underwent first BAV at our Institution between July 2007 and December 2012. Of these, the 202 patients (31.2%) who underwent BAV as bridge-to-decision (BTD) requiring clinical re-evaluation represented our study population. BTD patients were further subdivided in 5 groups: low left ventricular ejection fraction; mitral regurgitation grade ≥3; frailty; hemodynamic instability; comorbidity. The main objective of the study was to evaluate how BAV influenced the final treatment strategy in the whole BTD group and in its single specific subgroups. Results. Mean logistic EuroSCORE was 23.5±15.3%, mean age was 81±7 years. Mean transaortic gradient decreased from 47±17 mmHg to 33±14 mmHg. Of the 193 patients with BTD-BAV who received a second heart team evaluation, 72.5% were finally deemed eligible for definitive treatment (25.4%for AVR; 47.2% for TAVI): respectively, 96.7% of patients with left ventricular ejection fraction recovery; 70.5% of patients with mitral regurgitation reduction; 75.7% of patients who underwent BAV in clinical hemodynamic instability; 69.2% of frail patients and 68% of patients who presented relevant comorbidities. 27.5% of the study population was deemed ineligible for definitive treatment and treated with standard therapy/repeated BAV. In-hospital mortality was 4.5%, cerebrovascular accident occurred in 1% and overall vascular complications were 4% (0.5% major; 3.5% minor). Conclusions. Balloon aortic valvuloplasty should be considered as bridge-to-decision in high-risk patients with severe aortic stenosis who cannot be immediate candidates for definitive percutaneous or surgical treatment.

PEGylation and multimerization of the anti-p185HER-2 single chain Fv fragment 4D5: effects on tumor targeting

A major goal in antibody design for cancer therapy is to tailor the pharmacokinetic properties of the molecule according to specific treatment requirements. Key parameters determining the pharmacokinetics of therapeutic antibodies are target specificity, affinity, stability, and size. Using the p185HER-2 (HER-2)-specific scFv 4D5 as model system, we analyzed how changes in molecular weight and valency independently affect antigen binding and tumor localization. By employing multimerization and PEGylation, four different antibody formats were generated and compared with the scFv 4D5. First, dimeric and tetrameric miniantibodies were constructed by fusion of self-associating, disulfide-linked peptides to the scFv 4D5. Second, we attached a 20-kDa PEG moiety to the monovalent scFv and to the divalent miniantibody at the respective C terminus. In all formats, serum stability and full binding reactivity of the scFv 4D5 were retained. Functional affinity, however, did change. An avidity increase was achieved by multimerization, whereas PEGylation resulted in a 5-fold decreased affinity. Nevertheless, the PEGylated monomer showed an 8.5-fold, and the PEGylated dimer even a 14.5-fold higher tumor accumulation than the corresponding scFv, 48 h post-injection, because of a significantly longer serum half-life. In comparison, the non-PEGylated bivalent and tetravalent miniantibodies showed only a moderate increase in tumor localization compared with the scFv, which correlated with the degree of multimerization. However, these non-PEGylated formats resulted in higher tumor-to-blood ratios. Both multimerization and PEGylation represent thus useful strategies to tailor the pharmacokinetic properties of therapeutic antibodies and their combined use can additively improve tumor targeting.

Extracorporeal membrane oxygenation as a bridge to lung transplantation

The occurrence of severe graft failure after lung transplantation which appears refractory to conventional treatment represents a difficult situation with regard to the therapeutic strategies available. Of 17 patients undergoing single lung transplantation at our center, 2 developed early graft failure. In both, temporary artificial cardiopulmonary support by means of extracorporeal membrane oxygenation became necessary as a bridge to retransplantation. Both patients were successfully retransplanted after 8 h and 232 h, respectively, of extra-corporeal support. Postoperatively, there was a variety of complications. The first patient completely recovered from temporary severe cerebral dysfunction diagnosed as "locked-in syndrome". She was discharged from hospital on the 93rd postoperative day and remains alive and well 10 months after her operation. The other patient recovered well early after retransplantation. Later, however, airway problems developed, requiring the implantation of endotracheal stents. Cachexia and several episodes of viral pneumonia contributed to the progressive deterioration of her clinical status. She finally died after being hospitalized for 5 months after the original operation. These two cases illustrate the feasibility of using extracorporeal membrane oxygenation as a bridge to pulmonary transplantation.

On the layout of a least weight single span structure with uniform load. Some comments and improvements

Beghini et al (Struct Multidisc Optim doi:10.1007/s00158-013-1030-6, 2013) have published a very interesting paper arriving to practically the same nearly optimal solutions for the so named “bridge prob- lem” that the Writers published a year before, but using an alternative and remarkable approach to the problem. In spite of this general agreement, the Writers think that some details of the paper can be improved and there are results that can be given a clear and mean- ingful interpretation thanks to an old and practically unknown theorem on optimal slenderness.

Dynamics and folding of single two-stranded coiled-coil peptides studied by fluorescent energy transfer confocal microscopy

We report single-molecule measurements on the folding and unfolding conformational equilibrium distributions and dynamics of a disulfide crosslinked version of the two-stranded coiled coil from GCN4. The peptide has a fluorescent donor and acceptor at the N termini of its two chains and a Cys disulfide near its C terminus. Thus, folding brings the two N termini of the two chains close together, resulting in an enhancement of fluorescent resonant energy transfer. End-to-end distance distributions have thus been characterized under conditions where the peptide is nearly fully folded (0 M urea), unfolded (7.4 M urea), and in dynamic exchange between folded and unfolded states (3.0 M urea). The distributions have been compared for the peptide freely diffusing in solution and deposited onto aminopropyl silanized glass. As the urea concentration is increased, the mean end-to-end distance shifts to longer distances both in free solution and on the modified surface. The widths of these distributions indicate that the molecules are undergoing millisecond conformational fluctuations. Under all three conditions, these fluctuations gave nonexponential correlations on 1- to 100-ms time scale. A component of the correlation decay that was sensitive to the concentration of urea corresponded to that measured by bulk relaxation kinetics. The trajectories provided effective intramolecular diffusion coefficients as a function of the end-to-end distances for the folded and unfolded states. Single-molecule folding studies provide information concerning the distributions of conformational states in the folded, unfolded, and dynamically interconverting states.

The fifth epidermal growth factor-like domain of thrombomodulin does not have an epidermal growth factor-like disulfide bonding pattern.

The disulfide bonding pattern of the fourth and fifth epidermal growth factor (EGF)-like domains within the smallest active fragment of thrombomodulin have been determined. In previous work, this fragment was expressed and purified to homogeneity, and its cofactor activity, as measured by Kcat for thrombin activation of protein C, was the same as that for full-length thrombomodulin. CNBr cleavage at the single methionine in the connecting region between the domains and subsequent deglycosylation yielded the individual EGF-like domains. The disulfide bonds were mapped by partial reduction with tris(2-carboxyethyl)phosphine according to the method of Gray [Gray, W. R. (1993) Protein Sci. 2, 1732-1748], which provides unambiguous results. The disulfide bonding pattern of the fourth EGF-like domain was (1-3, 2-4, 5-6), which is the same as that found previously in EGF and in a synthetic version of the fourth EGF-like domain. Surprisingly, the disulfide bonding pattern of the fifth domain was (1-2, 3-4, 5-6), which is unlike that found in EGF or in any other EGF-like domain analyzed so far. This result is in line with an earlier observation that the (1-2, 3-4, 5-6) isomer bound to thrombin more tightly than the EGF-like (1-3, 2-4, 5-6) isomer. The observation that not all EGF-like domains have an EGF-like disulfide bonding pattern reveals an additional element of diversity in the structure of EGF-like domains.

English music (1604 to 1904) being the lectures given at the Music Loan Exhibition of the Worshipful Company of Musicians, held at Fishmongers' Hall, London Bridge, June-July, 1904.

The evolution of the pianoforte, by T.L. Southgate.- Our English songs, by W.H. Cummings.- The early English viols and their music, by H. Watson.- Madrigals, rounds, catches, glees, and part-songs, by E.M. Lee.- The recorder, flute, fife, and piccolo, by J. Finn.- Music in England in the year 1604, by Sir F. Bridge.- Our dances of bygone days, by A.S. Rose.- Masques and early operas, by A.H.D. Prendergast.- English opera after Purcell, by F.J. Sawyer.- Our cathedral composers and their works, by G.F. Huntley.- The single and double reed instruments, by D.J. Blaikley.- The water-organ of the ancients and the organ of to-day, by F.W. Galpin.- The regal and its successors: the harmonica, by T.L. Southgate.- The violin family and its music, by W.W. Cobbett.- The brass wind instruments, by J.E. Borland.- Some notes on early printed music, by A.H. Littleton.- Music of the country-side, by Sir E. Clarke.

Structural and functional characterization of the conserved salt bridge in mammalian Paneth cell alpha-defensins - Solution structures of mouse cryptdin-4 AND (E15D)-cryptdin-4

Defensins are mediators of mammalian innate immunity, and knowledge of their structure-function relationships is essential for understanding their mechanisms of action. We report here the NMR solution structures of the mouse Paneth cell α-defensin cryptdin-4 (Crp4) and a mutant (E15D)-Crp4 peptide, in which a conserved Glu15 residue was replaced by Asp. Structural analysis of the two peptides confirms the involvement of this Glu in a conserved salt bridge that is removed in the mutant because of the shortened side chain. Despite disruption of this structural feature, the peptide variant retains a well defined native fold because of a rearrangement of side chains, which result in compensating favorable interactions. Furthermore, salt bridge-deficient Crp4 mutants were tested for bactericidal effects and resistance to proteolytic degradation, and all of the variants had similar bactericidal activities and stability to proteolysis. These findings support the conclusion that the function of the conserved salt bridge in Crp4 is not linked to bactericidal activity or proteolytic stability of the mature peptide.