Penka Nikolova - Academia.edu (original) (raw)

Papers by Penka Nikolova

Biotechnol Bioeng, 1992

Whole cells of Saccharomyces cerevisiae analyzed the conversion of benzaldehyde to benzyl alcohol... more Whole cells of Saccharomyces cerevisiae analyzed the conversion of benzaldehyde to benzyl alcohol in aqueous-organic biphasic media. Reaction rate increased dramatically as moisture content of the solvent was increased in the range 0% to 2%. The highest biotransformation rates were observed when hexane was used as organic solvent. Benzaldehyde was also converted to benzyl alcohol by a cell-free crude extract in biphasic systems containing hexane, although the rate of product formation was much lower. Mutant strains of S. cerevisiae lacking some or all of the ADH isoenzymes, ADH I, II, and III, manifested similar rates for bioconversion of benzaldehyde to benzyl alcohol in both aqueous and two-phase systems. In general, conversion rates observed in aqueous media were 2 to 3 times higher than those observed in hexane containing 2% moisture.

Scientific Reports, 2016

The Melanoma-Associated Antigen A4 (MAGE-A4) protein is a target for cancer therapy. The function... more The Melanoma-Associated Antigen A4 (MAGE-A4) protein is a target for cancer therapy. The function of this protein is not well understood. We report the first comprehensive study on key cancer-associated MAGE-A4 mutations and provide analysis on the consequences of these mutations on the structure, folding and stability of the protein. Based on Nuclear Magnetic Resonance and Circular Dichroism, these mutations had no significant effects on the structure and the folding of the protein. Some mutations affected the thermal stability of the protein remarkably. Native mass spectrometry of wild-type MAGE-A4 showed a broad charge state distribution suggestive of a structurally dynamic protein. Significant intensity was found in relatively low charge states, indicative of a predominantly globular form and some population in more extended states. The latter is supported by Ion Mobility measurements. The MAGE-A4 mutants exhibited similar features. These novel molecular insights shed further light on better understanding of these proteins, which are implicated in a wide range of human cancers. Cancer/Testis (CT) antigens are a large family of proteins typically expressed in germ cells. CT antigens can also be overexpressed in an aberrant manner in various types of tumors such as melanoma, sarcoma, lung cancer, prostate cancer, breast cancer, ovarian cancer and a range of various other cancers 1-5. The first discovered CT antigen family member was melanoma-associated antigen A1 (MAGE-A1), which was identified as an antigen recognised by cytotoxic T lymphocytes in melanoma patients 6 , and was successfully cloned later 7,8. Since then, extensive chromosomal sequencing identified more than 50 genes of the MAGE family 9-18. Based on their expression pattern, the MAGE family can be divided into two subfamilies: type-I and type-II 19. While the expression of type-I subfamily is restricted to germ line and cancer cells, type-II subfamily is expressed in normal somatic tissues. Type-I MAGEs can be subdivided into MAGE-A,-B and-C groups. MAGE-A contains 12 genes (MAGE-A1 to-A12) where MAGE-A7 is a pseudogene 9. MAGE proteins consist of nearly 100 amino acid residues long N-terminal region, followed by two tandem winged helix domains with each wing termed as WH-A and WH-B, respectively 20. The N-terminal is rich in disorder promoting residues such as Ser, Pro, Glu and Arg, and thus this region is predicted to be disordered. The C-terminal region that spans over the two winged helix domains is highly conserved amongst type I and type II MAGEs and is known as MAGE Homology Domain (MHD). The crystal structure of MHD has been determined in a free state (MAGE-A4, PDB ID: 2AW0; MAGE-A3, PDB ID: 4V0P) and in complex with NSE1 (MAGE-G1, PDB ID: 3NW0) 20. The biological functions of MAGE proteins remain poorly understood. However, many reports correlate over-expression of type-I MAGEs with cancer malignance, tumor growth and poor patient prognosis. For example, MAGE-A2 was reported to promote tumor growth in normal oral keratinocytes and inhibits cell cycle arrest

Biochemistry Usa, Mar 1, 1997

The activity of the beta-1,4-glycanase Cex (EC 3.2.1.91) from Cellulomonas fimi is investigated i... more The activity of the beta-1,4-glycanase Cex (EC 3.2.1.91) from Cellulomonas fimi is investigated in connection with its industrial application in cellulose hydrolysis and its potential use in cellosaccharide synthesis. Catalytic activity measurements as a function of temperature, complemented with differential scanning calorimetry (DSC) data, are used to characterize the thermostability of the protein and the influence of interdomain interactions. The data suggest that the enzyme is irreversibly deactivated in one of two possible ways: (1) through a low-temperature route characterized by first-order kinetics; or (2) through a high-temperature route characterized by an initial reversible step followed by an irreversible step. Melting temperatures (Tm) of Cex and p-33 (the isolated catalytic domain of Cex) as estimated by DSC are 64.2 and 64.0 degrees C, respectively, suggesting that the binding and catalytic domains of the protein fold independently. Kinetic parameters (Km, kcat, and kcat/Km) of Cex for the hydrolysis of p-nitrophenyl beta-D-cellobioside (pNPC) were determined at temperatures ranging from 15 to 80 degrees C. As demanded by reversible mass-action thermodynamics, the Tm of Cex in the presence of excess ligand as determined from activity-temperature data is ca. 66.55 degrees C, more than 2 degrees C higher than the Tm for Cex under ligand-free conditions. The effect of temperature on the rate constant has been determined using Arrhenius plots. Combined with irreversible deactivation half-life data and DSC data, the results are used to evaluate a model, based on a theory developed by Hei et al. (1993), for predicting the time-dependent activity and active-state stability of the protein under a range of potential operating conditions.

Biocatalysis and Biotransformation, Jul 10, 2009

... Page 6. 334 2.0-1.5-PENKA NIKOLOVA AND OWEN P. WARD A . A A E E Y u 3 u 0 p. C 0 m - L 5 10-0... more ... Page 6. 334 2.0-1.5-PENKA NIKOLOVA AND OWEN P. WARD A . A A E E Y u 3 u 0 p. C 0 m - L 5 10-0 v) m L c) 0 0.5 - m - 00 ... nexane-Substrate Product ZX Rate, mM/h/g DW catalyst ma-Trifluoro-o-tolualdeh yde aaa-Trifluoro-rn-tolualde hyde ma-Trifluoro-p-tolualdeh yde ...

Annals of the New York Academy of Sciences

Biochemical Society Transactions

Abstract For proteins the link between their structure and their function is a principal tenet of... more Abstract For proteins the link between their structure and their function is a principal tenet of biology. The established approach to understand the function of a protein is to ‘solve’ its structure and subsequently investigate interactions between the protein and its binding partners. However, structure determination via crystallography or NMR is challenging for proteins where localised regions or even their entire structure fail to fold into a 3D structure. These so called intrinsically or inherently disordered proteins (IDPs) or intrinsically disordered regions (IDRs) constitute up to 40% of all expressed proteins, and a much higher percentage in proteins involved in the proliferation of cancer. For these proteins, there is a need to develop new methods for structure characterization which exploit their biophysical properties. Ion Mobility Mass Spectrometry is uniquely able to examine both absolute conformation(s), populations of conformation and also conformational change, and ...

International Journal of Mass Spectrometry

International Journal of Mass Spectrometry j o u r n a l h o m e p a g e : w w w . e l s e v i e ... more International Journal of Mass Spectrometry j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / i j m s Utilising ion mobility-mass spectrometry to interrogate macromolecules: Factor H complement control protein modules 10–15 and 19–20 and the DNA-binding core domain of tumour suppressor p53 a b s t r a c t Ion mobility-mass spectrometry (IM-MS) has been used to study the gas-phase structures of three proteins: the DNA-binding core domain of p53 (amino acid residues 94–312) and two fragments of com-plement control protein factor H (regions 10–15 and 19–20) which are involved in important cellular processes. We report collision cross-sections as a function of charge state for these systems, following electrospray ionisation from 'native' conditions. The DNA-binding core domain of p53 contains a zinc ion that is postulated to have an important role in retaining functionality. We have chelated the zinc from the protein using phenanthroline and observe...

Analytical chemistry, Jan 28, 2015

Thermally induced conformational transitions of three proteins of increasing intrinsic disorder, ... more Thermally induced conformational transitions of three proteins of increasing intrinsic disorder, cytochrome c, the tumour suppressor protein p53 DNA binding domain (p53 DBD) and the N-terminus of the oncoprotein Murine Double Minute2 (NT-MDM2), have been studied using near-native mass spectrometry and variable temperature drift time ion mobility mass spec-trometry (VT DT IM-MS). Ion mobility measurements were carried out at temperatures ranging from 200 K to 571 K. Multiple conformations are observable over several charge states for all three monomeric proteins, and for cytochrome c dimers of signifi-cant intensity are also observed. Cytochrome c [M+5H]5+ ions present in one conformer of CCS ~1200 Å2, undergoing compaction in line with the reported Tmelt = 360.15 K before slight unfolding at 571 K. The more extended [M+7H]7+ cytochrome c monomer presents as two conformers undergoing similar compaction and structural rearrangements, prior to thermally induced unfolding. The [D+11H]11...

The EMBO Journal, 2000

The core domain of p53 is extremely susceptible to mutations that lead to loss of function. We an... more The core domain of p53 is extremely susceptible to mutations that lead to loss of function. We analysed the stability and DNA-binding activity of such mutants to understand the mechanism of second-site suppressor mutations. Double-mutant cycles show that N239Y and N268D act as 'global stability' suppressors by increasing the stability of the cancer mutants G245S and V143A-the free energy changes are additive. Conversely, the suppressor H168R is specific for the R249S mutation: despite destabilizing wild type, H168R has virtually no effect on the stability of R249S, but restores its binding affinity for the gadd45 promoter. NMR structural comparisons of R249S/H168R and R249S/T123A/H168R with wild type and R249S show that H168R reverts some of the structural changes induced by R249S. These results have implications for possible drug therapy to restore the function of tumorigenic mutants of p53: the function of mutants such as V143A and G245S is theoretically possible to restore by small molecules that simply bind to and hence stabilize the native structure, whereas R249S requires alteration of its mutant native structure.

Proceedings of the National Academy of Sciences, 1998

We have designed a p53 DNA binding domain that has virtually the same binding affinity for the ga... more We have designed a p53 DNA binding domain that has virtually the same binding affinity for the gadd45 promoter as does wild-type protein but is considerably more stable. The design strategy was based on molecular evolution of the protein domain. Naturally occurring amino acid substitutions were identified by comparing the sequences of p53 homologues from 23 species, introducing them into wild-type human p53, and measuring the changes in stability. The most stable substitutions were combined in a multiple mutant. The advantage of this strategy is that, by substituting with naturally occurring residues, the function is likely to be unimpaired. All point mutants bind the consensus DNA sequence. The changes in stability ranged from ؉1.27 (less stable Q165K) to ؊1.49 (more stable N239Y) kcal mol ؊1 , respectively. The changes in free energy of unfolding on mutation are additive. Of interest, the two most stable mutants (N239Y and N268D) have been known to act as suppressors and restored the activity of two of the most common tumorigenic mutants. Of the 20 single mutants, 10 are cancerassociated, though their frequency of occurrence is extremely low: A129D, Q165K, Q167E, and D148E are less stable and M133L, V203A and N239Y are more stable whereas the rest are neutral. The quadruple mutant (M133LV203AN239YN-268D), which is stabilized by 2.65 kcal mol ؊1 and T m raised by 5.6°C is of potential interest for trials in vivo. The tumor suppressor protein p53 plays a key role in preventing the development of cancer (1, 2). The loss of p53 function, which results almost entirely from mutations within the DNA binding domain, is associated with half of all human cancers. Despite the potential therapeutic relevance, the p53 cellular stability and the mechanisms by which it is regulated are not well understood (3, 4, 5). It has been reported that p53 stability affects its functions, the most important of which are the cell-cycle arrest and apoptosis (4, 5, 6, 7). The cellular concentration of p53 protein is regulated tightly, and, in normal cells, it is kept at low levels through interactions with the Mdm2 protein. In response to stress signals, such as DNA damage, the interaction between the two proteins is compromised, which leads to a sharp increase of p53 concentration in the cell and in the half-life of the normal wild-type p53 protein (4, 5). Consequently, the accumulation of p53 protein in tumors is caused by an increase in the cellular stability of p53 (5). Several reports have highlighted the low thermal stability of p53, which may limit the cellular stability of p53 in the absence of Mdm2 (8, 9). In addition, it has been shown that some mutants of p53 have a wild-type-like conformation whereas others adopt an altered, ''mutant'' conformation, which is reported to affect function (10). Structural studies suggested that the mutant The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ''advertisement'' in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Proceedings of the National Academy of Sciences, 1997

Some 50% of human cancers are associated with mutations in the core domain of the tumor suppresso... more Some 50% of human cancers are associated with mutations in the core domain of the tumor suppressor p53. Many mutations are thought just to destabilize the protein. To assess this and the possibility of rescue, we have set up a system to analyze the stability of the core domain and its mutants. The use of differential scanning calorimetry or spectroscopy to measure its melting temperature leads to irreversible denaturation and aggregation and so is useful as only a qualitative guide to stability. There are excellent two-state denaturation curves on the addition of urea that may be analyzed quantitatively. One Zn 2؉ ion remains tightly bound in the holo-form of p53 throughout the denaturation curve. The stability of wild type is 6.0 kcal (1 kcal ‫؍‬ 4.18 kJ)͞mol at 25°C and 9.8 kcal͞mol at 10°C. The oncogenic mutants R175H, C242S, R248Q, R249S, and R273H are destabilized by 3.0, 2.9, 1.9, 1.9, and 0.4 kcal͞mol, respectively. Under certain denaturing conditions, the wildtype domain forms an aggregate that is relatively highly f luorescent at 340 nm on excitation at 280 nm. The destabilized mutants give this f luorescence under milder denaturation conditions.

Nucleic Acids Research, 2008

The apoptosis stimulating p53 proteins, ASPP1 and ASPP2, are the first two common activators of t... more The apoptosis stimulating p53 proteins, ASPP1 and ASPP2, are the first two common activators of the p53 protein family that selectively enable the latter to regulate specific apoptotic target genes, which facilitates yes yet unknown mechanisms for discrimination between cell cycle arrest and apoptosis. To better understand the interplay between ASPP-and p53-family of proteins we investigated the molecular interactions between them using biochemical methods and structure-based homology modelling. The data demonstrate that: (i) the binding of ASPP1 and ASPP2 to p53, p63 and p73 is direct; (ii) the C-termini of ASPP1 and ASPP2 interact with the DNA-binding domains of p53 protein family with dissociation constants, K d , in the lower micro-molar range; (iii) the stoichiometry of binding is 1:1; (iv) the DNA-binding domains of p53 family members are sufficient for these protein-protein interactions; (v) EMSA titrations revealed that while tri-complex formation between ASPPs, p53 family of proteins and PUMA/ Bax is mutually exclusive, ASPP2 (but not ASPP1) formed a complex with PUMA (but not Bax) and displaced p53 and p73. The structure-based homology modelling revealed subtle differences between ASPP2 and ASPP1 and together with the experimental data provide novel mechanistic insights.

Journal of Industrial Microbiology, 1994

ABSTRACT

Journal of Industrial Microbiology, 1993

In this paper biocatalytic reactions carried out by whole cells in nonconventional media are revi... more In this paper biocatalytic reactions carried out by whole cells in nonconventional media are reviewed. Similar relationships are observed between solvent hydrophobicity and catalytic activity in reactions carried out by isolated enzymes and whole cells. In addition to the effect of organic solvent on biocatalyst stability, microbial cells are susceptible to damaging effects caused by the organic phase. In general, more hydrophobic solvents manifest lower toxicity towards the cells. Whole cell biocatalysts require more water than isolated enzymes and two-phase systems have been most widely used to study whole cell biocatalysis. Immobilization makes cell biocatalysts more resistant to organic solvents and helps achieve homogeneous biocatalyst dispersion. Cell entrapment methods have been widely used with organic solvent systems and mixtures of natural and/or synthetic polymers allow adjustment of the hydrophobicity-hydrophilicity balance of the support matrix. Some examples of stereoselective catalysis using microbial cells in organic solvent media are presented.

Journal of Industrial Microbiology, 1992

Biotransformation ofbenzaldehyde and pyruvate to (R)-phenylacetyl carbinol by Saccharomyces cerev... more Biotransformation ofbenzaldehyde and pyruvate to (R)-phenylacetyl carbinol by Saccharomyces cerevisiae was investigated in two-phase aqueous-organic reaction media. With hexane as organic solvent, maximum biotransformation activity was observed with a moisture content of 10~o. Of the organic solvents tested, highest biotransformation activities were observed with hexane and hexadecane, and lowest activities occurred with chloroform and toluene. Biocatalyst samples from biphasic media containing hexane, decane and toluene manifested no apparent cell structural damage when examined using scanning electron microscopy. In contrast, cellular biocatalyst recovered from two-phase systems containing chloroform, butylacetate and ethylacetate exhibited damage in the form of cell puncturing after different incubation periods. Phospholipids were detected in reaction media from biocatalytic systems which exhibited cell damage in electron micrographs. Phospholipid release was much lower in the two-phase systems containing toluene or hexane or in 100% aqueous biocatalytic system.

Canadian Journal of Chemistry, 2000

We measured vapour pressures of aqueous acetonitrile (abbreviated as ACN) at 6, 20, and 37°C, fro... more We measured vapour pressures of aqueous acetonitrile (abbreviated as ACN) at 6, 20, and 37°C, from which excess chemical potentials of ACN (µ EACN) were calculated. We also determined excess partial molar enthalpies of ACN (H EACN) at 6, 20, 30, 37, and 45°C. From these data, excess partial molar entropies of ACN (S EACN) were calculated at 6, 20, and 37°C. Using density data by Benson's group, excess partial molar volumes of ACN (V EACN) were evaluated. The response function data by the same group were also used to evaluate amplitude and wavelength of mean-square fluctuations in terms of volume, entropy, and cross between volume and entropy. All the above quantities and their dependence on the mol fraction of solute, i.e., the effect of additional solute on the above quantities were used to study the effect of acetonitrile on the molecular organization of H2O. It was found that acetonitrile works as a stronger structure-making solute than methanol. Rather its effect on H2O is a...