Sharon Kelly

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Timir Tripathi

North-Eastern Hill University, Hill Univ. Shillong-793022 (India)

Ahmed A. Farghaly

Virginia Commonwealth University

Gobind Das

Istituto Italiano di Tecnologia / Italian Institute of Technology

Theivasanthi Thirugnanasambandan

Kalasalingam University

Atanas G Atanasov

Medical University of Vienna

Javier Ellena

Universidade de São Paulo

Harvinder Chawla

Govind Ballabh Pant Krishi evam Prodyogik Vishva Vidyalaya, Pantnagar, India

Diane M Bunce

The Catholic University of America

Yaffa Vitberg

Weill Cornell Medicine

Ilhami Kuru

Baskent University

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Papers by Sharon Kelly

Product-induced stabilization of tertiary and quaternary structure in Escherichia coli dehydroquinase

Journal of Biological Chemistry, 1994

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The Herpes Simplex Virus Triplex Protein, VP23, Exists as a Molten Globule

Journal of Virology, 1998

Two proteins, VP19C (50,260 Da) and VP23 (34,268 Da), make up the triplexes which connect adjacen... more Two proteins, VP19C (50,260 Da) and VP23 (34,268 Da), make up the triplexes which connect adjacent hexons and pentons in the herpes simplex virus type 1 capsid. VP23 was expressed in Escherichia coli and purified to homogeneity by Ni-agarose affinity chromatography. In vitro capsid assembly experiments demonstrated that the purified protein was functionally active. Its physical status was examined by differential scanning calorimetry, ultracentrifugation, size exclusion chromatography, circular dichroism, fluorescence spectroscopy, and 8-anilino-1-naphthalene sulfonate binding studies. These studies established that the bacterially expressed VP23 exhibits properties consistent with its being in a partially folded, molten globule state. We propose that the molten globule represents a functionally relevant intermediate which is necessary to allow VP23 to undergo interaction with VP19C in the process of capsid assembly.

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Characterisation of a pucBA deletion mutant from Rhodopseudomonas palustris lacking all but the pucBAd genes

Photosynthesis research, Jan 31, 2017

Rhodopseudomonas palustris is a species of purple photosynthetic bacteria that has a multigene fa... more Rhodopseudomonas palustris is a species of purple photosynthetic bacteria that has a multigene family of puc genes that encode the alpha and beta apoproteins, which form the LH2 complexes. A genetic dissection strategy has been adopted in order to try and understand which spectroscopic form of LH2 these different genes produce. This paper presents a characterisation of one of the deletion mutants generated in this program, the pucBAd only mutant. This mutant produces an unusual spectroscopic form of LH2 that only has a single large NIR absorption band at 800 nm. Spectroscopic and pigment analyses on this complex suggest that it has basically a similar overall structure as that of the wild-type HL LH2 complex. The mutant has the unique phenotype where the mutant LH2 complex is only produced when cells are grown at LL. At HL the mutant only produces the LH1-RC core complex.

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Studies on the unfolding and refolding of oligomeric proteins

The unfolding and refolding of a number of oligomeric enzymes have been studied. These were: fuma... more The unfolding and refolding of a number of oligomeric enzymes have been studied. These were: fumarase from pig heart, the NAD+-dependent isocitrate dehydrogenase from yeast, the citrate synthases from pig hean, Acinetobacter anitratum and Thermoplasma acidophilum and the chaperone protein GroEL from Escherichia coli. In each case the unfolding by guanidinium chloride (GdnHCI) was monitored by enzyme activity (to detect possible perturbations at the active site), protein fluorescence

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Probing Protein Sequences as Sources for Encrypted Antimicrobial Peptides

Plos One, 2012

Starting from the premise that a wealth of potentially biologically active peptides may lurk with... more Starting from the premise that a wealth of potentially biologically active peptides may lurk within proteins, we describe here a methodology to identify putative antimicrobial peptides encrypted in protein sequences. Candidate peptides were identified using a new screening procedure based on physicochemical criteria to reveal matching peptides within protein databases. Fifteen such peptides, along with a range of natural antimicrobial peptides, were examined using DSC and CD to characterize their interaction with phospholipid membranes. Principal component analysis of DSC data shows that the investigated peptides group according to their effects on the main phase transition of phospholipid vesicles, and that these effects correlate both to antimicrobial activity and to the changes in peptide secondary structure. Consequently, we have been able to identify novel antimicrobial peptides from larger proteins not hitherto associated with such activity, mimicking endogenous and/or exogenous microorganism enzymatic processing of parent proteins to smaller bioactive molecules. A biotechnological application for this methodology is explored. Soybean (Glycine max) plants, transformed to include a putative antimicrobial protein fragment encoded in its own genome were tested for tolerance against Phakopsora pachyrhizi, the causative agent of the Asian soybean rust. This procedure may represent an inventive alternative to the transgenic technology, since the genetic material to be used belongs to the host organism and not to exogenous sources.

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Transient supramolecular reconfiguration of peptide nanostructures using ultrasound

Materials Horizons, 2015

Ultrasound, i.e. high frequency oscillating pressure waves, was used to achieve transient reorgan... more

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Self-assembling multimeric integrin 5 1 ligands for cell attachment and spreading

Protein Engineering Design and Selection, 2008

Substrates utilising clustered arginine-glycine-aspartic acid (RGD) ligand displays support great... more Substrates utilising clustered arginine-glycine-aspartic acid (RGD) ligand displays support greater cell adhesion over random displays. However, cell adhesion to integrin a5b1 requires the synergy site on the 9th type III fibronectin domain (FIII) in addition to RGD on the 10th FIII domain. Here, we have designed and expressed soluble protein chimeras consisting of an N-terminal 9th-10th FIII domain pair, IgG-derived hinge and leucine zipper-derived helix; the latter mutated to yield di-, tri-and tetrameric coiled coils and thus self-assembling, multimeric integrin a5b1 ligands. A unique C-terminal cysteine was appended to the helix to facilitate 'anchoring' of the chimeras with a defined orientation on a surface. Size-exclusion chromatography and circular dichroism demonstrated that the chimeras self-assembled as multimers in solution with defined secondary structures predicted from theoretical calculations. Biotinylation via a thioether bond was used to selectively bind the chimeras to streptavidin-coated surfaces, each of which was then shown to bind integrin a5b1 by surface plasmon resonance. Spreading of fibroblasts to surfaces derivatised with the chimeras was found to proceed in the order: tetramer > trimer > dimer > monomer. Thus, we describe novel polyvalent integrin a5b1 ligands for facile derivatisation of substrates to improve cell adhesion in vitro.

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Conformational changes induced by DNA binding of NF-κB

Nucleic Acids Research, 1995

The transcription factor NF-icB makes extensive contacts with its recognition site over one compl... more The transcription factor NF-icB makes extensive contacts with its recognition site over one complete tum of the double helix. Structural transitions, in both protein and DNA, that accompany formation of the DNA-protein complex were analysed by proteinase sensitivity and circular dichroism (CD) spectroscopy. In the absence of DNA chymotrypsin cleaved p50 after residues Y60 and N78, while proteinase K cleaved p50 after residues S74 and Q180. Previous experiments had indicated that trypsin cleaved p50 after K77. Cleavages after Y60, S74, K77 and N78 were blocked in the presence of bound DNA, whereas cleavage after Q180 was enhanced. Y60, S74, K77 and N78 are all located in the p50 N-terminal domain AB loop, whereas Q180 is located in the mainly a-helical region between p50 N-terminal domain ,B-strands G' and H. As only Y60 makes direct contact with the DNA it is likely that the AB loop is highly unstructured in the absence of DNA, but is held in a rigid, proteinase-resistant structure by bound DNA. These conclusions were supported by CD spectroscopic studies of recombinant p50 and p65 homodimers, which indicated that both species changed conformion when binding DNA. Examination of the near UV CD spectra revealed tat wfth some DNA sequences the bound and free forms of the DNA assumed diferent onf ons. While this was evident for a fully symmetrical, high affinity recogniton site DNA, it was not apparent with less tightly bound DNA.

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Conformation of the Mineralocorticoid Receptor N-terminal Domain: Evidence for Induced and Stable Structure

Molecular Endocrinology, 2010

The mineralocorticoid receptor (MR) binds the steroid hormones aldosterone and cortisol and has a... more The mineralocorticoid receptor (MR) binds the steroid hormones aldosterone and cortisol and has an important physiological role in the control of salt homeostasis. Regions of the protein important for gene regulation have been mapped to the amino-terminal domain (NTD) and termed activation function (AF)1a, AF1b, and middle domain (MD). In the present study, we used a combination of biophysical and biochemical techniques to investigate the folding and function of the MR-NTD transactivation functions. We demonstrate that MR-AF1a and MR-MD have relatively little stable secondary structure but have the propensity to form α-helical conformation. Induced folding of the MR-MD enhanced protein-protein binding with a number of coregulatory proteins, including the coactivator cAMP response element-binding protein-binding protein and the corepressors SMRT and RIP140. By contrast, the MR-AF1b domain appeared to have a more stable conformation consisting predominantly of β-secondary structure. F...

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Secondary structures of narcissus mosaic virus coat protein

Journal of General Virology, 1991

Circular dichroism (CD) measurements on the coat protein of narcissus mosaic virus particles show... more

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Mutational Analysis of Phototropin 1 Provides Insights into the Mechanism Underlying LOV2 Signal Transmission

Journal of Biological Chemistry, 2006

Phototropins (phot1 and phot2) are blue light-activated serine/threonine protein kinases that eli... more Phototropins (phot1 and phot2) are blue light-activated serine/threonine protein kinases that elicit a variety of photoresponses in plants. Light sensing by the phototropins is mediated by two flavin mononucleotide (FMN)-binding domains, designated LOV1 and LOV2, located in the N-terminal region of the protein. Exposure to light results in the formation of a covalent adduct between the FMN chromophore and a conserved cysteine residue within the LOV domain. LOV2 photoexcitation is essential for phot1 function in Arabidopsis and is necessary to activate phot1 kinase activity through light-induced structural changes within a conserved ␣-helix situated C-terminal to LOV2. Here we have used site-directed mutagenesis to identify further amino acid residues that are important for phot1 activation by light. Mutagenesis of bacterially expressed LOV2 and full-length phot1 expressed in insect cells indicates that perturbation of the conserved salt bridge on the surface of LOV2 does not play a role in receptor activation. However, mutation of a conserved glutamine residue (Gln 575) within LOV2, reported previously to be required to propagate structural changes at the LOV2 surface, attenuates light-induced autophosphorylation of phot1 expressed in insect cells without compromising FMN binding. These findings, in combination with double mutant analyses, indicate that Gln 575 plays an important role in coupling light-driven cysteinyl adduct formation from within LOV2 to structural changes at the LOV2 surface that lead to activation of the C-terminal kinase domain.

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The Ov20 Protein of the Parasitic Nematode Onchocerca volvulus

Journal of Biological Chemistry, 1997

Ov20 is a major antigen of the parasitic nematode Onchocerca volvulus, the causative agent of riv... more Ov20 is a major antigen of the parasitic nematode Onchocerca volvulus, the causative agent of river blindness in humans, and the protein is secreted into the tissue occupied by the parasite. DNA encoding Ov20 was isolated, and the protein was expressed in Escherichia coli. Fluorescence-based ligand binding assays show that the protein contains a high affinity binding site for retinol, fluorescent fatty acids (11-((5-dimethylaminonaphthalene-1-sulfonyl)amino)undecanoic acid, dansyl-DL-␣-aminocaprylic acid, and parinaric acid) and, by competition, oleic and arachidonic acids, but not cholesterol. The fluorescence emission of dansylated fatty acids is significantly blue-shifted upon binding in comparison to similarly sized ␤-sheet-rich mammalian retinoland fatty acid-binding proteins. Secondary structure prediction algorithms indicate that a ␣-helix predominates in Ov20, possibly in a coiled coil motif, with no evidence of ␤ structures, and this was confirmed by circular dichroism. The protein is highly stable in solution, requiring temperatures in excess of 90°C or high denaturant concentrations for unfolding. Ov20 therefore represents a novel class of small retinol-binding protein, which appears to be confined to nematodes. The retinol binding activity of Ov20 could possibly contribute to the eye defects associated with onchocerciasis and, because there is no counterpart in mammals, represents a strategic target for chemotherapy.

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The aromatic amino acid content of the bacterial chaperone protein groEL (cpn60) Evidence for the presence of a single tryptophan

FEBS Letters, 1991

Studies of the absorption and fluorescence properties of tile chaperone protein groEL (cpn60) fro... more Studies of the absorption and fluorescence properties of tile chaperone protein groEL (cpn60) from £scherichia coil show that tryptophan is present. in contrast to the proposed amino acid sequence of the protein (Hemmingsen, S.M. et al. 0988) Nature 333, 330-334). By detemlining a suitable value for the specific absorption coefficient of the protein at 280 nm, it has been shown that the content of the aromatic amino acids corresponds to a single tryptophan and (most probably) seven tyrosines per subunit (Mr 57 200).

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Structural plasticity of the feline leukaemia virus fusion peptide: a circular dichroism study

FEBS Letters, 1998

The secondary structure of the feline leukaemia virus (FeLV) fusion peptide was investigated usin... more The secondary structure of the feline leukaemia virus (FeLV) fusion peptide was investigated using circular dichroism (CD). Our results show that this peptide can readily flip between random, K K-helical and L L-sheet conformations, depending upon its environment. The CD spectrum changes from one characteristic of random coil to predominantly L L-sheet type, and finally to that showing the characteristics of K K-helical structure on moving from an aqueous solvent, through several increasingly hydrophobic systems, to a highly hydrophobic solvent. Electron microscopy confirmed the presence of L L structure. We propose that the structural plasticity demonstrated here is crucial to the ability of the fusion peptide to perturb lipid bilayers, and thus promote membrane fusion.

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Co-operativity between modules within a C3b-binding site of complement receptor type 1

FEBS Letters, 1999

Complement receptor type 1 (CR1) has 30 modules in its extracellular portion. An understanding of... more Complement receptor type 1 (CR1) has 30 modules in its extracellular portion. An understanding of structure-function relationships within CR1 is being assembled gradually from studies of overlapping protein fragments. A CR1 fragment corresponding to modules 16 and 17 was expressed recombinantly as a non-glycosylated protein and its stability and unfolding characteristics studied using biophysical techniques. The results were compared with data collected previously on a CR1 fragment encompassing modules 15, 16 and 17 which together constitute a C3b-binding site (Kirkitadze, M.

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The secondary structure of influenza A M2 transmembrane domain A circular dichroism study

FEBS Letters, 1992

Using circular diehroism, this study investigated the secondary structure of the influenza A M2 t... more Using circular diehroism, this study investigated the secondary structure of the influenza A M2 transmembrane domain. When reconstituted into 1,2-dioleoyl-stl-glyc~ro-3-phosphecholine liposolneS, the M2 transmembrane domain was found to adopt a predominantly a-helical secondary structure which was unaffected by both temperature and the addition of l-aminoadamantane hydrochloride. Reeonstitution into 1,2.dioleoyl-snglycero.3.phosphoglycerol liposomes resulted in a marked decrease in helical content.

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Tight junction modulation and biochemical characterisation of the zonula occludens toxin C-and N-termini

FEBS Letters, 2007

The ZOT N-terminal domain was expressed and refolded, yielding a soluble protein with defined sec... more The ZOT N-terminal domain was expressed and refolded, yielding a soluble protein with defined secondary structure. Although distantly related to protein I of filamentous phages, no evidence of ATPase activity was found. It is therefore unlikely that the ZOT N-terminal domain is involved in cholera toxin phage packaging in Vibrio cholerae. The ZOT C-terminal domain caused delocalisation of occludin and ZO-1 from Caco-2 cell-cell contacts, irrespective of disulfide bridge formation in its putative binding domain. However, the C-terminal domain did not cause actin reorganisation and this may explain the absence of a concomitant reduction in the transepithelial electrical resistance across cell monolayers.

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The Use of Circular Dichroism in the Investigation of Protein Structure and Function

Current Protein & Peptide Science, 2000

Circular Dichroism (CD) relies on the differential absorption of left and right circularly polari... more Circular Dichroism (CD) relies on the differential absorption of left and right circularly polarised radiation by chromophores which either possess intrinsic chirality or are placed in chiral environments. Proteins possess a number of chromophores which can give rise to CD signals. In the far UV region (240-180 nm), which corresponds to peptide bond absorption, the CD spectrum can be analysed to give the content of regular secondary structural features such as α-helix and β-sheet. The CD spectrum in the near UV region (320-260 nm) reflects the environments of the aromatic amino acid side chains and thus gives information about the tertiary structure of the protein. Other non-protein chromophores such as flavin and haem moieties can give rise to CD signals which depend on the precise environment of the chromophore concerned. Because of its relatively modest resource demands, CD has been used extensively to give useful information about protein structure, the extent and rate of structural changes and ligand binding. In the protein design field, CD is used to assess the structure and stability of the designed protein fragments. Studies of protein folding make extensive use of CD to examine the folding pathway; the technique has been especially important in characterising "molten globule" intermediates which may be involved in the folding process. CD is an extremely useful technique for assessing the structural integrity of membrane proteins during extraction and characterisation procedures. The interactions between chromophores can give rise to characteristic CD signals. This is well illustrated by the case of the light harvesting complex from photosynthetic bacteria, where the CD spectra can be analysed to indicate the extent of orbital overlap between the rings of bacteriochlorophyll molecules. It is therefore evident that CD is a versatile technique in structural biology, with an increasingly wide range of applications.

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Optical Spectroscopic Methods for Probing the Conformational Stability of Immobilised Enzymes

ChemPhysChem, 2009

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Incorporation of N-heterocyclic cations into proteins with a highly directed chemical modification

Chemical Communications, 2007

N-Heterocyclic cations are incorporated into proteins using 5-(2-bromoethyl)phenanthridinium brom... more N-Heterocyclic cations are incorporated into proteins using 5-(2-bromoethyl)phenanthridinium bromide, which selectively reacts with either cysteine or lysine residues, resulting in ethylphenanthridinium (Phen) or highly stable cyclised dihydro-imidazo-phenanthridinium (DIP) adducts respectively; these modifications have been found to manipulate the observed structure of lysozyme and bovine serum albumin by AFM.

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Product-induced stabilization of tertiary and quaternary structure in Escherichia coli dehydroquinase

Journal of Biological Chemistry, 1994

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The Herpes Simplex Virus Triplex Protein, VP23, Exists as a Molten Globule

Journal of Virology, 1998

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Characterisation of a pucBA deletion mutant from Rhodopseudomonas palustris lacking all but the pucBAd genes

Photosynthesis research, Jan 31, 2017

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Studies on the unfolding and refolding of oligomeric proteins

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