Significance of bound water to local chain conformations in protein crystals.

We examine how the polypeptide chain in protein crystal structures exploits the multivalent hydrogen-bonding potential of bound water molecules. This shows that multiple interactions with a single water molecule tend to occur locally along the chain. A distinctive internal-coordinate representation of the local water-binding segments reveals several consensus conformations. The fractional water occupancy of each was found by comparison of the total number of conformations in the database regardless of the presence or absence of bound water. The water molecule appears particularly frequently in type II beta-turn geometries and an N-terminal helix feature. This work constitutes a first step into assessing not only the generality but also the significance of specific water binding in globular proteins.

Computer determination of peptide conformations in water: different roads to structure.

Fragments of proteins (short peptides) that "fold" suggest a mechanism of how complete conformational search in protein folding is avoided. We used a computational method to determine structures of two foldable peptides in explicit water: RVEW and CSVTC. The optimization starts from random structures and no experimental constraints are used. In agreement with NMR data, the simulations find a hydrophobic pair (Val/Trp) in REVW. The structure of CSVTC is induced by a surface water that bridges two amide hydrogens, a drive to structure hypothesized by Ben-Naim [Ben-Naim, A. (1990) J. Chem. Phys. 93, 8196-8210] that is largely ignored in studies of folding. Tendency to structure in short peptide chains suggests a mechanism for the formation of short-range nucleation sites in protein folding.

Metal Clips Induce Folding of a Short Unstructured Peptide into an a-Helix via Turn Conformations in Water. Kinetic versus Thermodynamic Products

Short peptides corresponding to two to four a-helical turns of proteins are not thermodynamically stable helices in water. Unstructured octapeptide Ac-His1*-Ala2-Ala3-His4*-His5*-Glu6-Leu7-His8*-NH2 (1) reacts with two [Pd ((NH2)-N-15(CH2)(2) (NH2)-N-15)(NO3)(2)] in water to form a kinetically stable intermediate, [{Pden}(2)-{(1,4)(5,8)-peptide}](2), in which two 19-membered metallocyclic rings stabilize two peptide turns. Slow subsequent folding to a thermodynamically more stable two-turn a-helix drives the equilibrium to [{Pden}(2)-{(1,5)(4,8)-peptide}] (3), featuring two 22-membered rings. This transformation from unstructured peptide via turns to an a-helix suggests that metal clips might be useful probes for investigating peptide folding.

Mechanical actuation by responsive polyelectrolyte brushes and triblock gels

Progress in the development of actuating molecular devices based on responsive polymers is reviewed. The synthesis and characterization of "grafted from brushes and triblock copolymers is reported. The responsive nature of polyelectrolyte brushes, grown by surface initiated atomic transfer radical polymerization (ATRP), has been characterized by scanning force microscopy, neutron reflectometry, and single molecule force measurements. The molecular response is measured directly for the brushes in terms of both the brush height and composition and the force generated by a single molecule. Triblock copolymers, based on hydrophobic end blocks and polyacid midblock, have been used to produce polymer gels where the deformation of the molecules can be followed directly by small angle Xray scattering (SAXS), and a correlation between molecular shape change and macroscopic deformation has been established. A Landolt pHoscillator, based on bromate/sulfite/ferrocyanide, with a room temperature period of 20 min and a range of 3.1 polyelectrolyte molecules scale affinely to produce the macroscopic response of the system in an oscillating chemical reaction.

The relationship between charge density and polyelectrolyte brush profile using simultaneous neutron reflectivity and in situ attenuated total internal reflection FTIR

We report on a novel experimental study of a pH-responsive polyelectrolyte brush at the silicon/D2O interface. A poly[2-(diethylamino)ethyl methacrylate] brush was grown on a large silicon crystal which acted as both a substrate for a neutron reflectivity solid/liquid experiment but also as an FTIR-ATR spectroscopy crystal. This arrangement has allowed for both neutron reflectivities and FTIR spectroscopic information to be measured in parallel. The chosen polybase brush shows strong IR bands which can be assigned to the N-D+ stretch, D2O, and a carbonyl group. From such FTIR data, we are able to closely monitor the degree of protonation along the polymer chain as well as revealing information concerning the D2O concentration at the interface. The neutron reflectivity data allows us to determine the physical brush profile normal to the solid/liquid interface along with the corresponding degree of hydration. This combined approach makes it possible to quantify the charge on a polymer brush alongside the morphology adopted by the polymer chains. © 2013 American Chemical Society.

PREPARATION OF POLYELECTROLYTE MEMBRANES EMBEDDED WITH ZEOLITE NANOPARTICLES FOR ENHANCED PERFORMANCE IN FORWARD OSMOSIS

Water scarcity is a global issue that has already affected every continent. Membrane technology is considered as one of the most promising candidates for resolving this worsening issue. Among all the membrane processes, the emerging forward osmosis (FO) membrane process is osmotically-driven and has unique advantages compared with other traditional pressure-driven membrane processes. One of the major challenges to advancing the FO membrane process is the lack of a suitable membrane. Polyelectrolyte thin film prepared via layer-by-layer (LbL) technique has demonstrated its excellent performance in many applications including electronics, optics, sensors, etc. Recent studies have revealed the potential of polyelectrolyte thin films in acting as the active separation layer of FO membranes, but significant efforts are still needed to improve the membrane performance and understand the transport mechanisms. This dissertation introduces a novel approach to prepare a zeolite-embedded polyelectrolyte composite membrane for enhanced FO performance. This membrane takes advantages of the versatile LbL process to unprecedentedly incorporate high loading of zeolite nanoparticles, which are anticipated to facilitate water transport due to the uniquely interconnected structure of zeolites. Major topics discussed in this dissertation include: (1) the synthesis and evaluation of the polyelectrolyte-zeolite composite FO membrane, (2) the examination of the fouling resistance to identify its technical limitations, (3) the demonstration of the membrane regenerability as an effective strategy for membrane fouling control, and (4) the investigation of crosslinking effects on the membrane performance to elucidate the transport mechanisms involved in the zeolite-embedded polyelectrolyte membranes. Comparative studies have been made between polyelectrolyte membranes with and without zeolite incorporation. The findings suggest that the zeolite-embedded membrane, although slightly more susceptible to silica scaling, has demonstrated enhanced water flux and separation capability, good resistance to organic fouling, and complete regenerability for fouling control. Additionally, the embedded zeolite nanoparticles are proved to be able to create fast pathways for water transport. Overall, this work provides a novel strategy to create zeolite-polymer composite membranes with enhanced separation performance and unique fouling mitigation properties.

Alternate Conformations Regulate Ribosomal Recoding in a Positive-sense RNA Virus

Positive-sense RNA viruses are important animal, plant, insect and bacteria pathogens and constitute the largest group of RNA viruses. Due to the relatively small size of their genomes, these viruses have evolved a variety of non-canonical translation mechanisms to optimize coding capacity expanding their proteome diversity. One such strategy is codon redefinition or recoding. First described in viruses, recoding is a programmed translation event in which codon alterations are context dependent. Recoding takes place in a subset of messenger RNA (mRNAs) with some products reflecting new, and some reflecting standard, meanings. The ratio between the two is both critical and highly regulated. While a variety of recoding mechanisms have been documented, (ribosome shunting, stop-carry on, termination-reinitiation, and translational bypassing), the two most extensively employed by RNA viruses are Programmed Ribosomal Frameshifting (PRF) and Programmed Ribosomal Readthrough (PRT). While both PRT and PRF subvert normal decoding for expression of C-terminal extension products, the former involves an alteration of reading frame, and the latter requires decoding of a non-sense codon. Both processes occur at a low but defined frequency, and both require Recoding Stimulatory Elements (RSE) for regulation and optimum functionality. These stimulatory signals can be embedded in the RNA in the form of sequence or secondary structure, or trans-acting factors outside the mRNA such as proteins or micro RNAs (miRNA). Despite 40+ years of study, the precise mechanisms by which viral RSE mediate ribosome recoding for the synthesis of their proteins, or how the ratio of these products is maintained, is poorly defined. This study reveals that in addition to a long distance RNA:RNA interaction, three alternate conformations and a phylogenetically conserved pseudoknot regulate PRT in the carmovirus Turnip crinkle virus (TCV).

Charged pullulan derivatives for the development of nanocarriers by polyelectrolyte complexation

Pullulan, a neutral polysaccharide, was chemically modified in order to obtain two charged derivatives: reaction with SO3(.)DMF complex afforded a sulfate derivative (SP), while reaction with glycidyltrimethylammonium chloride gave a quaternary ammonium salt (AP). The presence of the charged groups was confirmed by FTIR. Assessment of the positions where the reaction took place was based on (1)H- and (13)C NMR (COSY, HSQC-TOCSY, HSQC-DEPT, and HMBC) experiments. Estimation of the degree of substitution (DS) was made from elemental analysis data, and further confirmed by NMR peak areas in the case of AP. These new derivatives showed the capability to condense with each other, forming nanoparticles with the ability to associate a model protein (BSA) and displaying adequate size for drug delivery applications, therefore making them good candidates for the production of pullulan-based nanocarriers by polyelectrolyte complexation.

Vibrational spectroscopic studies of wool

Fourier transfonn (FT) Raman, Raman microspectroscopy and Fourier transform infrared (FTIR) spectroscopy have been used for the structural analysis and characterisation of untreated and chemically treated wool fibres. For FT -Raman spectroscopy novel methods of sample presentation have been developed and optimised for the analysis of wool. No significant fluorescence was observed and the spectra could be obtained routinely. The stability of wool keratin to the laser source was investigated and the visual and spectroscopic signs of sample damage were established. Wool keratin was found to be extremely robust with no signs of sample degradation observed for laser powers of up to 600 m W and for exposure times of up to seven and half hours. Due to improvements in band resolution and signal-to-noise ratio, several previously unobserved spectral features have become apparent. The assignment of the Raman active vibrational modes of wool have been reviewed and updated to include these features. The infrared spectroscopic techniques of attenuated total reflectance (ATR) and photoacoustic (P A) have been used to examine shrinkproofed and mothproofed wool samples. Shrinkproofing is an oxidative chemical treatment used to selectively modifY the surface of a wool fibre. Mothproofing is a chemical treatment applied to wool for the prevention of insect attack. The ability of PAS and A TR to vary the penetration depth by varying certain instrumental parameters was used to obtain spectra of the near surface regions of these chemically treated samples. These spectra were compared with those taken with a greater penetration depth, which therefore represent more of the bulk wool sample. The PA and ATR spectra demonstrated that oxidation was restricted to the near-surface layer of wool. Extensive curve fitting of ATR spectra of untreated wool indicated that cuticle was composed of a mixed protein conformation, but was predominately that of an a.-helix. The cortex was proposed to be a mixture of both a.helical and ~-pleated sheet protein conformations. These findings were supported by PAS depth profiling results. Raman microspectroscopy was used in an extensive investigation of the molecular structure of the wool fibre. This included determining the orientation of certain functional groups within the wool fibre and the symmetry of particular vibrations. The orientation ofbonds within the wool fibre was investigated by orientating the wool fibre axis parallel and then perpendicular to the plane of polarisation of the electric vector of the incident radiation. It was experimentally determined that the majority of C=O and N-H bonds of the peptide bond of wool lie parallel to the fibre axis. Additionally, a number of the important vibrations associated with the a-helix were also found to lie parallel to the fibre axis. Further investigation into the molecular structure of wool involved determining what effect stretching the wool fibre had on bond orientation. Raman spectra of stretched and unstretched wool fibres indicated that extension altered the orientation ofthe aromatic rings, the CH2 and CH3 groups of the amino acids. Curve fitting results revealed that extension resulted in significant destruction of the a-helix structure a substantial increase in the P-pleated sheet structure. Finally, depolarisation ratios were calculated for Raman spectra. The vibrations associated with the aromatic rings of amino acids had very low ratios which indicated that the vibrations were highly symmetrical.

Rational design of serine protease inhibitors

Proteases regulate a spectrum of diverse physiological processes, and dysregulation of proteolytic activity drives a plethora of pathological conditions. Understanding protease function is essential to appreciating many aspects of normal physiology and progression of disease. Consequently, development of potent and specific inhibitors of proteolytic enzymes is vital to provide tools for the dissection of protease function in biological systems and for the treatment of diseases linked to aberrant proteolytic activity. The studies in this thesis describe the rational design of potent inhibitors of three proteases that are implicated in disease development. Additionally, key features of the interaction of proteases and their cognate inhibitors or substrates are analysed and a series of rational inhibitor design principles are expounded and tested. Rational design of protease inhibitors relies on a comprehensive understanding of protease structure and biochemistry. Analysis of known protease cleavage sites in proteins and peptides is a commonly used source of such information. However, model peptide substrate and protein sequences have widely differing levels of backbone constraint and hence can adopt highly divergent structures when binding to a protease’s active site. This may result in identical sequences in peptides and proteins having different conformations and diverse spatial distribution of amino acid functionalities. Regardless of this, protein and peptide cleavage sites are often regarded as being equivalent. One of the key findings in the following studies is a definitive demonstration of the lack of equivalence between these two classes of substrate and invalidation of the common practice of using the sequences of model peptide substrates to predict cleavage of proteins in vivo. Another important feature for protease substrate recognition is subsite cooperativity. This type of cooperativity is commonly referred to as protease or substrate binding subsite cooperativity and is distinct from allosteric cooperativity, where binding of a molecule distant from the protease active site affects the binding affinity of a substrate. Subsite cooperativity may be intramolecular where neighbouring residues in substrates are interacting, affecting the scissile bond’s susceptibility to protease cleavage. Subsite cooperativity can also be intermolecular where a particular residue’s contribution to binding affinity changes depending on the identity of neighbouring amino acids. Although numerous studies have identified subsite cooperativity effects, these findings are frequently ignored in investigations probing subsite selectivity by screening against diverse combinatorial libraries of peptides (positional scanning synthetic combinatorial library; PS-SCL). This strategy for determining cleavage specificity relies on the averaged rates of hydrolysis for an uncharacterised ensemble of peptide sequences, as opposed to the defined rate of hydrolysis of a known specific substrate. Further, since PS-SCL screens probe the preference of the various protease subsites independently, this method is inherently unable to detect subsite cooperativity. However, mean hydrolysis rates from PS-SCL screens are often interpreted as being comparable to those produced by single peptide cleavages. Before this study no large systematic evaluation had been made to determine the level of correlation between protease selectivity as predicted by screening against a library of combinatorial peptides and cleavage of individual peptides. This subject is specifically explored in the studies described here. In order to establish whether PS-SCL screens could accurately determine the substrate preferences of proteases, a systematic comparison of data from PS-SCLs with libraries containing individually synthesised peptides (sparse matrix library; SML) was carried out. These SML libraries were designed to include all possible sequence combinations of the residues that were suggested to be preferred by a protease using the PS-SCL method. SML screening against the three serine proteases kallikrein 4 (KLK4), kallikrein 14 (KLK14) and plasmin revealed highly preferred peptide substrates that could not have been deduced by PS-SCL screening alone. Comparing protease subsite preference profiles from screens of the two types of peptide libraries showed that the most preferred substrates were not detected by PS SCL screening as a consequence of intermolecular cooperativity being negated by the very nature of PS SCL screening. Sequences that are highly favoured as result of intermolecular cooperativity achieve optimal protease subsite occupancy, and thereby interact with very specific determinants of the protease. Identifying these substrate sequences is important since they may be used to produce potent and selective inhibitors of protolytic enzymes. This study found that highly favoured substrate sequences that relied on intermolecular cooperativity allowed for the production of potent inhibitors of KLK4, KLK14 and plasmin. Peptide aldehydes based on preferred plasmin sequences produced high affinity transition state analogue inhibitors for this protease. The most potent of these maintained specificity over plasma kallikrein (known to have a very similar substrate preference to plasmin). Furthermore, the efficiency of this inhibitor in blocking fibrinolysis in vitro was comparable to aprotinin, which previously saw clinical use to reduce perioperative bleeding. One substrate sequence particularly favoured by KLK4 was substituted into the 14 amino acid, circular sunflower trypsin inhibitor (SFTI). This resulted in a highly potent and selective inhibitor (SFTI-FCQR) which attenuated protease activated receptor signalling by KLK4 in vitro. Moreover, SFTI-FCQR and paclitaxel synergistically reduced growth of ovarian cancer cells in vitro, making this inhibitor a lead compound for further therapeutic development. Similar incorporation of a preferred KLK14 amino acid sequence into the SFTI scaffold produced a potent inhibitor for this protease. However, the conformationally constrained SFTI backbone enforced a different intramolecular cooperativity, which masked a KLK14 specific determinant. As a consequence, the level of selectivity achievable was lower than that found for the KLK4 inhibitor. Standard mechanism inhibitors such as SFTI rely on a stable acyl-enzyme intermediate for high affinity binding. This is achieved by a conformationally constrained canonical binding loop that allows for reformation of the scissile peptide bond after cleavage. Amino acid substitutions within the inhibitor to target a particular protease may compromise structural determinants that support the rigidity of the binding loop and thereby prevent the engineered inhibitor reaching its full potential. An in silico analysis was carried out to examine the potential for further improvements to the potency and selectivity of the SFTI-based KLK4 and KLK14 inhibitors. Molecular dynamics simulations suggested that the substitutions within SFTI required to target KLK4 and KLK14 had compromised the intramolecular hydrogen bond network of the inhibitor and caused a concomitant loss of binding loop stability. Furthermore in silico amino acid substitution revealed a consistent correlation between a higher frequency of formation and the number of internal hydrogen bonds of SFTI-variants and lower inhibition constants. These predictions allowed for the production of second generation inhibitors with enhanced binding affinity toward both targets and highlight the importance of considering intramolecular cooperativity effects when engineering proteins or circular peptides to target proteases. The findings from this study show that although PS-SCLs are a useful tool for high throughput screening of approximate protease preference, later refinement by SML screening is needed to reveal optimal subsite occupancy due to cooperativity in substrate recognition. This investigation has also demonstrated the importance of maintaining structural determinants of backbone constraint and conformation when engineering standard mechanism inhibitors for new targets. Combined these results show that backbone conformation and amino acid cooperativity have more prominent roles than previously appreciated in determining substrate/inhibitor specificity and binding affinity. The three key inhibitors designed during this investigation are now being developed as lead compounds for cancer chemotherapy, control of fibrinolysis and cosmeceutical applications. These compounds form the basis of a portfolio of intellectual property which will be further developed in the coming years.

Homo- and heteronuclear meso,meso-(E)-Ethene-1,2-diyl-Linked Diporphyrins : preparation, X-ray crystal structure, electronic absorption and emission spectra and density functional theory calculations

Homo-and heteronuclear meso,meso-(E)-ethene-1,2-diyl-linked diporphyrins have been prepared by the Suzuki coupling of porphyrinylboronates and iodovinylporphyrins. Combinations comprising 5,10,15-triphenylporphyrin (TriPP) on both ends of the ethene-1,2-diyl bridge M 210 (M 2=H 2/Ni, Ni 2, Ni/Zn, H 4, H 2Zn, Zn 2) and 5,15-bis(3,5-di-tert-butylphenyl)porphyrinato-nickel(II) on one end and H 2, Ni, and ZnTriPP on the other (M 211), enable the first studies of this class of compounds possessing intrinsic polarity. The compounds were characterized by electronic absorption and steady state emission spectra, 1H NMR spectra, and for the Ni 2 bis(TriPP) complex Ni 210, single crystal X-ray structure determination. The crystal structure shows ruffled distortions of the porphyrin rings, typical of Ni II porphyrins, and the (E)-C 2H 2 bridge makes a dihedral angle of 50° with the mean planes of the macrocycles. The result is a stepped parallel arrangement of the porphyrin rings. The dihedral angles in the solid state reflect the interplay of steric and electronic effects of the bridge on interporphyrin communication. The emission spectra in particular, suggest energy transfer across the bridge is fast in conformations in which the bridge is nearly coplanar with the rings. Comparisons of the fluorescence behaviour of H 410 and H 2Ni10 show strong quenching of the free base fluorescence when the complex is excited at the lower energy component of the Soret band, a feature associated in the literature with more planar conformations. TDDFT calculations on the gas-phase optimized geometry of Ni 210 reproduce the features of the experimental electronic absorption spectrum within 0.1 eV. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Bis(benzylaminium) 4,5-dichlorobenzene-1,2-dicarboxylate monohydrate

In the structure of the title salt 2C7H10N+.C8H2Cl2O4(2-) .H2O the two benzylaminium anions have different conformations, one being essentially planar the other having the side-chain rotated out of the benzene plane (minimum ring to side-chain C-C-C-N torsion angles = -3.6(6) and 50.1(5)\%, respectively). In the 4,5-dichlorophthalate dianion the carboxyl groups make ihedral angles of 23.0(2) and 76.5(2)\% with the benzene ring. Aminium N-H...O and water O-H...O hydrogen-bonding associations with carboxyl O-atom acceptors give a two-dimensional duplex sheet structure which extends along the (011) plane. Weak pi-pi interactions are also present between the benzene ring and one of the cation rings [minimum ring centroid separation = 3.749(3)Ang.

Structure of the immature retroviral capsid at 8 Å resolution by cryo-electron microscopy

The assembly of retroviruses such as HIV-1 is driven by oligomerization of their major structural protein, Gag. Gag is a multidomain polyprotein including three conserved folded domains: MA (matrix), CA (capsid) and NC (nucleocapsid)(1). Assembly of an infectious virion proceeds in two stages(2). In the first stage, Gag oligomerization into a hexameric protein lattice leads to the formation of an incomplete, roughly spherical protein shell that buds through the plasma membrane of the infected cell to release an enveloped immature virus particle. In the second stage, cleavage of Gag by the viral protease leads to rearrangement of the particle interior, converting the non-infectious immature virus particle into a mature infectious virion. The immature Gag shell acts as the pivotal intermediate in assembly and is a potential target for anti-retroviral drugs both in inhibiting virus assembly and in disrupting virus maturation(3). However, detailed structural information on the immature Gag shell has not previously been available. For this reason it is unclear what protein conformations and interfaces mediate the interactions between domains and therefore the assembly of retrovirus particles, and what structural transitions are associated with retrovirus maturation. Here we solve the structure of the immature retroviral Gag shell from Mason-Pfizer monkey virus by combining cryo-electron microscopy and tomography. The 8-angstrom resolution structure permits the derivation of a pseudo-atomic model of CA in the immature retrovirus, which defines the protein interfaces mediating retrovirus assembly. We show that transition of an immature retrovirus into its mature infectious form involves marked rotations and translations of CA domains, that the roles of the amino-terminal and carboxy-terminal domains of CA in assembling the immature and mature hexameric lattices are exchanged, and that the CA interactions that stabilize the immature and mature viruses are almost completely distinct.

Superactivation of metal electrode surfaces and its relevance to COads oxidation at fuel cell anodes

The inhibiting effect of COads on platinum-based anodes is a major problem in the development of ambient temperature, polyelectrolyte membrane-type fuel cells. One of the unusual features of the response for the oxidative removal of the species in question is that the response observed for this reaction in the positive sweep is highly dependent on the CO admission potential, for example, when the COads is formed in the Hads region it undergoes oxidation at unusually low potentials. Such behaviour is attributed here to hydrogen activation of the platinum surface, with the result that oxide mediators (and COads oxidation) occurs at an earlier stage of the positive sweep. It is also demonstrated, for both platinum and gold in acid solution, that dramatic premonolayer oxidation responses may be observed following suitable preactivation of the electrode surfaces. It is suggested that the defect state of a solid electrode surface is an important variable whose investigation may yield improved fuel cell anode performance.