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Heat and SDS insensitive NDK dimers are largely stabilised by hydrophobic interaction to form functional hexamer in Mycobacterium smegmatis

100%

Arumugam M., Ajitkumar P.

Acta Biochimica Polonica

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2013

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tom 60

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nr 2

The primary structure and function of nucleoside diphosphate kinase (NDK), a substrate non-specific enzyme involved in the maintenance of nucleotide pools is also implicated to play pivotal roles in many other cellular processes. NDK is conserved from bacteria to human and forms a homotetramer or hexamer to exhibit its biological activity. However, the nature of the functional oligomeric form of the enzyme differs among different organisms. The functional form of NDKs from many bacterial systems, including that of the human pathogen, Mycobacterium tuberculosis (MtuNDK), is a hexamer, although some bacterial NDKs are tetrameric in nature. The present study addresses the oligomeric property of MsmNDK and how a dimer, the basic subunit of a functional hexamer, is stabilized by hydrogen bonds and hydrophobic interactions. Homology modeling was generated using the three-dimensional structure of MtuNDK as a template; the residues interacting at the monomer-monomer interface of MsmNDK were mapped. Using recombinant enzymes of wild type, catalytically inactive mutant, and monomer-monomer interactive mutants of MsmNDK, the stability of the dimer was verified under heat, SDS, low pH, and methanol. The predicted residues (Gln17, Ser24 and Glu27) were engaged in dimer formation, however the mutated proteins retained the ATPase and GTPase activity even after introducing single (MsmNDK- Q17A, MsmNDK-E27A, and MsmNDK-E27Q) and double (MsmNDK-E27A/Q17A) mutation. However, the monomer-monomer interaction could be abolished using methanol, indicating the stabilization of the monomer-monomer interaction by hydrophobic interaction.

2

Changes of quantities of tocopherols and their dimers during storage of soya-bean oil under N2 and under air

100%

Gogolewski M., Galuba G., Bartkowiak-Fludra E., Jasinska-Stepniak A.

Food Science and Technology. Scientific Papers of Agricultural University of Poznan

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2000

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tom 04

3

Crystal structure of the alpha-actinin rod: four spectrin repeats forming a tight dimer

100%

Ylanne J., Scheffzek K., Young P., Saraste M.

Cellular and Molecular Biology Letters

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2001

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tom 06

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nr 2

4

Mutational analysis of the AtNUDT7 Nudix hydrolase from Arabidopsis thaliana reveals residues required for protein quarternary structure formation and activity

84%

Olejnik K., Plochocka D., Grynberg M., Goch G., Gruszecki W. I., Basinska T., Kraszewska E.

Acta Biochimica Polonica

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2009

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tom 56

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nr 2

291-300

Arabidopsis thaliana AtNUDT7, a homodimeric Nudix hydrolase active on ADP-ribose and NADH, exerts negative control on the major signaling complex involved in plant defense activation and programmed cell death. The structural and functional consequences of altering several amino-acid residues of the AtNUDT7 protein have been examined by site-directed mutagenesis, far-UV circular dichroism (CD), attenuated total reflection-Fourier transform infrared (ATR-FTIR) and photon correlation (PCS) spectroscopy, biochemical analysis and protein-protein interaction studies. Alanine substitutions of F73 and V168 disallowed dimer formation. Both the F73A- and V168A-mutated proteins displayed no observable enzymatic activity. Alanine substitution of the V69 residue did not significantly alter the enzyme activity and had no influence on dimer arrangement. The non-conserved V26 residue, used as a negative control, did not contribute to the enzyme quaternary structure or activity. Detailed biophysical characterization of the wild-type and mutant proteins indicates that the mutations do not considerably alter the secondary structure of the enzyme but they affect dimer assembly. In addition, mutating residues V69, F73 and V168 disrupted the binding of AtNUDT7 to the regulatory 14.3.3 protein. These are the first studies of the structure-function relationship of AtNUDT7, a Nudix hydrolase of important regulatory function.

5

Kinetic intermediates of unfolding of dimeric prostatic phosphatase

84%

Kuciel R., Mazurkiewicz A., Dudzik P.

Acta Biochimica Polonica

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2007

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tom 54

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nr 2

Kinetics of guanidine hydrochloride (GdnHCl)-induced unfolding of human prostatic acid phosphatase (hPAP), a homodimer of 50 kDa subunit molecular mass was investigated with enzyme activity measurements, capacity for binding an external hydrophobic probe, 1-anilinonaphtalene-8-sulfonate (ANS), accessibility of thiols to reaction with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and 2-(4'-maleimidylanilino)naphthalene-6-sulfonate (MIANS) and ability to bind Congo red dye. Kinetic analysis was performed to describe a possible mechanism of hPAP unfolding and dissociation that leads to generation of an inactive monomeric intermediate that resembles, in solution of 1.25 M GdnHCl pH 7.5, at 20°C, in equilibrium, a molten globule state. The reaction of hPAP inactivation in 1.25 M GdnHCl followed first order kinetics with the reaction rate constant 0.0715 ± 0.0024 min-1. The rate constants of similar range were found for the pseudo-first-order reactions of ANS and Congo red binding: 0.0366 ± 0.0018 min-1 and 0.0409 ± 0.0052 min-1, respectively. Free thiol groups, inaccessible in the native protein, were gradually becoming, with the progress of unfolding, exposed for the reactions with DTNB and MIANS, with the pseudo-first-order reaction rate constants 0.327 ± 0.014 min-1 and 0.216 ± 0.010 min-1, respectively. The data indicated that in the course of hPAP denaturation exposure of thiol groups to reagents took place faster than the enzyme inactivation and exposure of the protein hydrophobic surface. This suggested the existence of a catalytically active, partially unfolded, but probably dimeric kinetic intermediate in the process of hPAP unfolding. On the other hand, the protein inactivation was accompanied by exposure of a hydrophobic, ANS-binding surface, and with an increased capacity to bind Congo red. Together with previous studies these results suggest that the stability of the catalytically active conformation of the enzyme depends mainly on the dimeric structure of the native hPAP.

6

Overproduction and purification of the CepA protein from Lactococcus lactis

84%

Kowalczyk M., Bardowski J.

Acta Biochimica Polonica

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2003

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tom 50

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nr 2

In this work we present cloning and overexpression of lactococcal CcpA protein in Escherichia coli Xllblue strain as a fusion with 6xHis tag. A high yield of the CcpA protein was obtained when the cells were cultured in liquid medium LB with 100 ug/ml ampicillin at 37°C and subsequently for 4 h after induction by IPTG. The procedure let us obtain 5 mg of homogenous CcpA protein. Glutaraldehyde crosslinking analysis indicated the formation of dimer or tetramer forms of the CcpA protein.

7

Preparation of alpha-, gamma-, delta-tocopherol dimers and their analysis with HPLC

84%

Galuba G. A., Gogolewski M., Jasinska-Stepniak A.

Food Science and Technology. Scientific Papers of Agricultural University of Poznan

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1998

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tom 02

Ograniczanie wyników

Heat and SDS insensitive NDK dimers are largely stabilised by hydrophobic interaction to form functional hexamer in Mycobacterium smegmatis

Changes of quantities of tocopherols and their dimers during storage of soya-bean oil under N2 and under air

Crystal structure of the alpha-actinin rod: four spectrin repeats forming a tight dimer

Mutational analysis of the AtNUDT7 Nudix hydrolase from Arabidopsis thaliana reveals residues required for protein quarternary structure formation and activity

Kinetic intermediates of unfolding of dimeric prostatic phosphatase

Overproduction and purification of the CepA protein from Lactococcus lactis

Preparation of alpha-, gamma-, delta-tocopherol dimers and their analysis with HPLC