Structural basis of an embryonically lethal single Ala → Thr mutation in the vnd/NK-2 homeodomain

The structural and DNA binding behavior is described for an analog of the vnd/NK-2 homeodomain, which contains a single amino acid residue alanine to threonine replacement in position 35 of the homeodomain. Multidimensional nuclear magnetic resonance, circular dichroism, and electrophoretic gel retardation assays were carried out on recombinant 80-aa residue proteins that encompass the wild-type and mutant homeodomains. The mutant A35T vnd/NK-2 homeodomain is unable to adopt a folded conformation free in solution at temperatures down to −5°C in contrast to the behavior of the corresponding wild-type vnd/NK-2 homeodomain, which is folded into a functional three-dimensional structure below 25°C. The A35T vnd/NK-2 binds specifically to the vnd/NK-2 target DNA sequence, but with an affinity that is 50-fold lower than that of the wild-type homeodomain. Although the three-dimensional structure of the mutant A35T vnd/NK-2 in the DNA bound state shows characteristic helix–turn–helix behavior similar to that of the wild-type homeodomain, a notable structural deviation in the mutant A35T analog is observed for the amide proton of leucine-40. The wild-type homeodomain forms an unusual i,i-5 hydrogen bond with the backbone amide oxygen of residue 35. In the A35T mutant this amide proton resonance is shifted upfield by 1.27 ppm relative to the resonance frequency for the wild-type analog, thereby indicating a significant alteration of this i,i-5 hydrogen bond.

Structural studies of p21Waf1/Cip1/Sdi1 in the free and Cdk2-bound state: conformational disorder mediates binding diversity.

The cyclin-dependent kinase (Cdk) inhibitor p21Waf1/Cip1/Sdi1, important for p53-dependent cell cycle control, mediates G1/S arrest through inhibition of Cdks and possibly through inhibition of DNA replication. Cdk inhibition requires a sequence of approximately 60 amino acids within the p21 NH2 terminus. We show, using proteolytic mapping, circular dichroism spectropolarimetry, and nuclear magnetic resonance spectroscopy, that p21 and NH2-terminal fragments that are active as Cdk inhibitors lack stable secondary or tertiary structure in the free solution state. In sharp contrast to the disordered free state, however, the p21 NH2 terminus adopts an ordered stable conformation when bound to Cdk2, as shown directly by NMR spectroscopy. We have, thus, identified a striking disorder-order transition for p21 upon binding to one of its biological targets, Cdk2. This structural transition has profound implications in light of the ability of p21 to bind and inhibit a diverse family of cyclin-Cdk complexes, including cyclin A-Cdk2, cyclin E-Cdk2, and cyclin D-Cdk4. Our findings suggest that the flexibility, or disorder, of free p21 is associated with binding diversity and offer insights into the role for structural disorder in mediating binding specificity in biological systems. Further, these observations challenge the generally accepted view of proteins that stable secondary and tertiary structure are prerequisites for biological activity and suggest that a broader view of protein structure should be considered in the context of structure-activity relationships.

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.

Chemical synthesis and structure elucidation of bovine kappa-casein (1-44)

The caseins (alpha(s1), alpha(s2), beta, and kappa) are phosphoproteins present in bovine milk that have been studied for over a century and whose structures remain obscure. Here we describe the chemical synthesis and structure elucidation of the N-terminal segment (1-44) of bovine K-casein, the protein which maintains the micellar structure of the caseins. K-Casein (1-44) was synthesised by highly optimised Boc solid-phase peptide chemistry and characterised by mass spectrometry. Structure elucidation was carried out by circular dichroism and nuclear magnetic resonance spectroscopy. CD analysis demonstrated that the segment was ill defined in aqueous medium but in 30% trifluoroethanol it exhibited considerable helical structure. Further, NMR analysis showed the presence of a helical segment containing 26 residues which extends from Pro(8) to Arg(34). This is the first report which demonstrates extensive secondary structure within the casein class of proteins. (c) 2006 Elsevier Inc. All rights reserved.

Asymmetric reactions of organosilicon compounds and properties of novel optically active organosilanes

Partial reduction of racemic methoxysilanes by 1:1 complexes of lithium aluminium hydride with optically active cinchona and ephedra alkaloids give optically active silanes and methoxysilanes. Optical yields depend on the groups attached to silicon and the alkaloid used but in some cases approach 50%, The method has been used to prepare novel optically active organosilanes, possessing an asymmetric silicon centre, which are either inaccessible by any of the other available routes or would require a time consuming preparation. Such compounds are of use in the study of the mechanism of substitutions at silicon. Attempts have been made to rationalize the results of the asymmetric reductions in terms of differences in sterio and electronic interactions in diastereoisomeric transition states. Circular dichroism and optical rotatory dispersion spectra have been obtained for the optically active products in an attempt to elucidate the absolute configurations of the novel asymmetric organosilanes. The results from these studies provide a useful addition to the data so far accumulated for asymmetrically perturbed aromatic chromophores. Nuclear magnetic resonanoe studies of diastereoisomaric (-)-menthoxysilanes show that these compounds possess resonances extremely useful in the determination of optical purities for asymmetric organosilanes which possess an aromatic group. The effect of variable temperature on the spectra has revealed evidence for the conformational preferences in these compounds. Other diastereoisomeric alkoxysilanes have been prepared and their n.m.r.spectra studied in the hope of establishing trends. Exploratory studies for other asymmetric reactions proceeding at silicon have proved unfruitful.

Synthesis and Biological Evaluation of Novel Folic Acid Receptor-Targeted, β-Cyclodextrin-Based Drug Complexes for Cancer Treatment

Drug targeting is an active area of research and nano-scaled drug delivery systems hold tremendous potential for the treatment of neoplasms. In this study, a novel cyclodextrin (CD)-based nanoparticle drug delivery system has been assembled and characterized for the therapy of folate receptor-positive [FR(+)] cancer. Water-soluble folic acid (FA)-conjugated CD carriers (FACDs) were successfully synthesized and their structures were confirmed by 1D/2D nuclear magnetic resonance (NMR), matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS), high performance liquid chromatography (HPLC), Fourier transform infrared spectroscopy (FTIR), and circular dichroism. Drug complexes of adamatane (Ada) and cytotoxic doxorubicin (Dox) with FACD were readily obtained by mixed solvent precipitation. The average size of FACD-Ada-Dox was 1.5–2.5 nm. The host-guest association constant Ka was 1,639 M−1 as determined by induced circular dichroism and the hydrophilicity of the FACDs was greatly enhanced compared to unmodified CD. Cellular uptake and FR binding competitive experiments demonstrated an efficient and preferentially targeted delivery of Dox into FR-positive tumor cells and a sustained drug release profile was seen in vitro. The delivery of Dox into FR(+) cancer cells via endocytosis was observed by confocal microscopy and drug uptake of the targeted nanoparticles was 8-fold greater than that of non-targeted drug complexes. Our docking results suggest that FA, FACD and FACD-Ada-Dox could bind human hedgehog interacting protein that contains a FR domain. Mouse cardiomyocytes as well as fibroblast treated with FACD-Ada-Dox had significantly lower levels of reactive oxygen species, with increased content of glutathione and glutathione peroxidase activity, indicating a reduced potential for Dox-induced cardiotoxicity. These results indicate that the targeted drug complex possesses high drug association and sustained drug release properties with good biocompatibility and physiological stability. The novel FA-conjugated β-CD based drug complex might be promising as an anti-tumor treatment for FR(+) cancer.

Structure and lipid interactions of membrane-associated glycosyltransferases : Cationic patches and anionic lipids regulate biomembrane binding of both GT-A and GT-B enzymes

This thesis concerns work on structure and membrane interactions of enzymes involved in lipid synthesis, biomembrane and cell wall regulation and cell defense processes. These proteins, known as glycosyltransferases (GTs), are involved in the transfer of sugar moieties from nucleotide sugars to lipids or chitin polymers. Glycosyltransferases from three types of organisms have been investigated; one is responsible for vital lipid synthesis in Arabidopsis thaliana (atDGD2) and adjusts the lipid content in biomembranes if the plant experiences stressful growth conditions. This enzyme shares many structural features with another GT found in gram-negative bacteria (WaaG). WaaG is however continuously active and involved in synthesis of the protective lipopolysaccharide layer in the cell walls of Escherichia coli. The third type of enzymes investigated here are chitin synthases (ChS) coupled to filamentous growth in the oomycete Saprolegnia monoica. I have investigated two ChS-derived MIT domains that may be involved in membrane interactions within the endosomal pathway. From analysis of the three-dimensional structure and the amino-acid sequence, some important regions of these very large proteins were selected for in vitro studies. By the use of an array of biophysical methods (e.g. Nuclear Magnetic Resonance, Fluorescence and Circular Dichroism spectroscopy) and directed sequence analyses it was possible to shed light on some important details regarding the structure and membrane-interacting properties of the GTs. The importance of basic amino-acid residues and hydrophobic anchoring segments, both generally and for the abovementioned proteins specifically, is discussed. Also, the topology and amino-acid sequence of GT-B enzymes of the GT4 family are analyzed with emphasis on their biomembrane association modes. The results presented herein regarding the structural and lipid-interacting properties of GTs aid in the general understanding of glycosyltransferase activity. Since GTs are involved in a high number of biochemical processes in vivo it is of outmost importance to understand the underlying processes responsible for their activity, structure and interaction events. The results are likely to be useful for many applications and future experimental design within life sciences and biomedicine.

Chiroptical spectroscopy of biomolecules using chiral plasmonic nanostructures

This thesis explores the potential of chiral plasmonic nanostructures for the ultrasensitive detection of protein structure. These nanostructures support the generation of fields with enhanced chirality relative to circularly polarised light and are an extremely incisive probe of protein structure. In chapter 4 we introduce a nanopatterned Au film (Templated Plasmonic Substrate, TPS) fabricated using a high through-put injection moulding technique which is a viable alternative to expensive lithographically fabricated nanostructures. The optical and chiroptical properties of TPS nanostructures are found to be highly dependent on the coupling between the electric and magnetic modes of the constituent solid and inverse structures. Significantly, refractive index based measurements of strongly coupled TPSs display a similar sensitivity to protein structure as previous lithographic nanostructures. We subsequently endeavour to improve the sensing properties of TPS nanostructures by developing a high through-put nanoscale chemical functionalisation technique. This process involves a chemical protection/deprotection strategy. The protection step generates a self-assembled monolayer (SAM) of a thermally responsive polymer on the TPS surface which inhibits protein binding. The deprotection step exploits the presence of nanolocalised thermal gradients in the water surrounding the TPS upon irradiation with an 8ns pulsed laser to modify the SAM conformation on surfaces with high net chirality. This allows binding of biomaterial in these regions and subsequently enhances the TPS sensitivity levels. In chapter 6 an alternative method for the detection of protein structure using TPS nanostructures is introduced. This technique relies on mediation of the electric/magnetic coupling in the TPS by the adsorbed protein. This phenomenon is probed through both linear reflectance and nonlinear second harmonic generation (SHG) measurements. Detection of protein structure using this method does not require the presence of fields of enhanced chirality whilst it is also sensitive to a larger array of secondary structure motifs than the measurements in chapters 4 and 5. Finally, a preliminary investigation into the detection of mesoscale biological structure is presented. Sensitivity to the mesoscale helical pitch of insulin amyloid fibrils is displayed through the asymmetry in the circular dichroism (CD) of lithographic gammadions of varying thickness upon adsorption of insulin amyloid fibril spherulites and fragmented fibrils. The proposed model for this sensitivity to the helical pitch relies on the vertical height of the nanostructures relative to this structural property as well as the binding orientation of the fibrils.

Electro-spinning of pure collagen nano-fibres – just an expensive way to make gelatin?

Scaffolds manufactured from biological materials promise better clinical functionality, providing that characteristic features are preserved. Collagen, a prominent biopolymer, is used extensively for tissue engineering applications, because its signature biological and physico-chemical properties are retained in vitro preparations. We show here for the first time that the very properties that have established collagen as the leading natural biomaterial are lost when it is electro-spun into nano-fibres out of fluoroalcohols such as 1,1,1,3,3,3-hexafluoro-2-propanol or 2,2,2-trifluoroethanol. We further identify the use of fluoroalcohols as the major culprit in the process. The resultant nano-scaffolds lack the unique ultra-structural axial periodicity that confirms quarter-staggered supramolecular assemblies and the capacity to generate second harmonic signals, representing the typical crystalline triple-helical structure. They were also characterised by low denaturation temperatures, similar to those obtained from gelatin preparations ( p > 0.05). Likewise, circular dichroism spectra revealed extensive denaturation of the electro-spun collagen. Using pepsin digestion in combination with quantitative SDS-PAGE, we corroborate great losses of up to 99% of triple-helical collagen. In conclusion, electro-spinning of collagen out of fluoroalcohols effectively denatures this biopolymer, and thus appears to defeat its purpose, namely to create biomimetic scaffolds emulating the collagen structure and function of the extracellular matrix.

Evaluation of novel Streptococcus pyogenes vaccine candidates incorporating multiple conserved sequences from the C-repeat region of the M-protein

A major challenge for Streptococcus pyogenes vaccine development is the identification of epitopes that confer protection from infection by multiple S. pyogenes M-types. Here we have identified and characterised the distribution of common variant sequences from individual repeat units of the C-repeat region (CRR) of M-proteins representing 77 different M-types. Three polyvalent fusion vaccine candidates (SV1, SV2 and SV3) incorporating the most common variants were subsequently expressed and purified, and demonstrated to be alpha-helical by Circular Dichroism (CD), a secondary conformational characteristic of the CRR in the M-protein. Antibodies raised against each of these constructs recognise M-proteins that vary in their CRR, and bind to the surface of multiple S. pyogenes isolates. Antibodies raised against SV1, containing five variant sequences, also kill heterologous S. pyogenes isolates in in vitro bactericidal assays. Further structural characterisation of this construct demonstrated the conformation of SV1 was stable at different pHs, and thermal unfolding of SV1 a reversible process. Our findings demonstrate that linkage of multiple variant sequences into a single recombinant construct overcomes the need to embed the variant sequences in foreign helix promoting flanking sequences for conformational stability, and demonstrates the viability of the polyvalent candidates as global S. pyogenes vaccine candidates.

Analysis of complex molecular systems: The impact of multivariate analysis for resolving the interactions of small molecules with biopolymers - a review

This review is focused on the impact of chemometrics for resolving data sets collected from investigations of the interactions of small molecules with biopolymers. These samples have been analyzed with various instrumental techniques, such as fluorescence, ultraviolet–visible spectroscopy, and voltammetry. The impact of two powerful and demonstrably useful multivariate methods for resolution of complex data—multivariate curve resolution–alternating least squares (MCR–ALS) and parallel factor analysis (PARAFAC)—is highlighted through analysis of applications involving the interactions of small molecules with the biopolymers, serum albumin, and deoxyribonucleic acid. The outcomes illustrated that significant information extracted by the chemometric methods was unattainable by simple, univariate data analysis. In addition, although the techniques used to collect data were confined to ultraviolet–visible spectroscopy, fluorescence spectroscopy, circular dichroism, and voltammetry, data profiles produced by other techniques may also be processed. Topics considered including binding sites and modes, cooperative and competitive small molecule binding, kinetics, and thermodynamics of ligand binding, and the folding and unfolding of biopolymers. Applications of the MCR–ALS and PARAFAC methods reviewed were primarily published between 2008 and 2013.

Spectroscopic studies of the interactions of the pyrethroid insecticide fenvalerate with gramicidin

Fenvalerate is a pyrethroid insecticide which interacts with ionic channels. Using circular dichroism technique we have studied the interaction of fenvalerate with gramicidin, a model channel peptide which transports ions. In most organic solvents, gramicidin exists as a double helix except in trifluoroethanol where it exists as a channel forming single stranded beta(6.3) helical monomer. In model lipid membranes, under certain experimental conditions, gramicidin exists as a channel forming single stranded beta(6.3) helical dimer. Our results show that fenvalerate interacts more with the single stranded beta(6.3) helical monomer or dimer than with the double helical form of gramicidin. This was further confirmed by an increase in the rate of gramicidin mediated proton transport in liposomes by fenvalerate, using the pH sensitive fluorophore, pyranine.

A New Class Of Photo-Cross-Linkable Side-Chain Liquid Crystalline Polymers Containing Bis(Benzylidene)Cyclohexanone Units

This paper reports a new class of photo-cross-linkable side chain liquid crystalline polymers (PSCLCPs) based on the bis(benzylidene)cyclohexanone unit, which functions as both a mesogen and a photoactive center. Polymers with the bis(benzylidene)cyclohexanone unit and varying spacer length have been synthesized. Copolymers of bis(benzylidene)cyclohexanone containing monomer and cholesterol benzoate containing monomer with different compositions have also been prepared. All these polymers have been structurally characterized by spectroscopic techniques. Thermal transitions were studied by DSC, and mesophases were identified by polarized light optical microscopy (POM). The intermediate compounds OH-x, the monomers SCLCM-x, and the corresponding polymers PSCLCP-x, which are essentially based on bis(benzylidene)cyclohexanone, all show a nematic mesophase. Transition temperatures were observed to decrease with increasing spacer length. The copolymers with varying compositions exhibit a cholesteric mesophase, and the transition temperatures increase with the cholesteric benzoate units in the copolymer. Photolysis of the low molecular weight liquid crystalline bis(benzylidene)-cyclohexanone compound reveals that there are two kinds of photoreactions in these systems: the EZ photoisomerization and 2 pi + 2 pi addition. The EZ photoisomerization in the LC phase disrupts the parallel stacking of the mesogens, resulting in the transition from the LC phase to the isotropic phase. The photoreaction involving the 2 pi + 2 pi addition of the bis(benzylidene)cyclohexanone units in the polymer results in the cross-linking of the chains. The liquid crystalline induced circular dichroism (LCICD) studies of the cholesterol benzoate copolymers revealed that the cholesteric supramolecular order remains even after the photo-cross-linking.

Helix-sheet interconversion in a synthetic pore lining peptide derived from a designed ion channel

We have designed a four-helix protein that is expected to tetramerize in the membrane to form an ion channel with a structurally well defined pore. A synthetic peptide corresponding to the channel lining helix facilitates ion transport across liposomal membranes and largely helical in membranes. Detailed circular dichroism studies of the peptide in methanol, water and methanal-water mixtures reveal that it is helical in methanol, beta-structured in 97.5% water and a combination of these two structures at intermediate compositions of methanol and water. A fluorescence resonance energy transfer study of the peptide shows that the peptide is monomeric in methanol but undergoes extensive anti-parallel aggregation in aqueous solution.