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BBA - Proteins and Proteomics (v.1751, #2)
How to study proteins by circular dichroism by Sharon M. Kelly; Thomas J. Jess; Nicholas C. Price (pp. 119-139).
Circular dichroism (CD) is being increasingly recognised as a valuable technique for examining the structure of proteins in solution. However, the value of many studies using CD is compromised either by inappropriate experimental design or by lack of attention to key aspects of instrument calibration or sample characterisation. In this article, we summarise the basis of the CD approach and its application to the study of proteins, and then present clear guidelines on how reliable data can be obtained and analysed.
Keywords: Circular dichroism; Protein structure; Secondary structure; Protein folding; Ligand binding
Kinetic isotope effects of nucleoside hydrolase from Escherichia coli by Cindy Hunt; Niloufar Gillani; Anthony Farone; Mansoureh Rezaei; Paul C. Kline (pp. 140-149).
rihC is one of a group of three ribonucleoside hydrolases found in Escherichia coli ( E. coli). The enzyme catalyzes the hydrolysis of selected nucleosides to ribose and the corresponding base. A family of Vmax/ Km kinetic isotope effects using uridine labeled with stable isotopes, such as2H,13C, and15N, were determined by liquid chromatography/mass spectrometry (LC/MS). The kinetic isotope effects were 1.012±0.006, 1.027±0.005, 1.134±0.007, 1.122±0.008, and 1.002±0.004 for [1′-13C], [1-15N], [1′-2H], [2′-2H], and [5′-2H2] uridine, respectively. A transition state based upon a bond-energy bond-order vibrational analysis (BEBOVIB) of the observed kinetic isotope effects is proposed. The main features of this transition state are activation of the heterocyclic base by protonation of/or hydrogen bonding to O2, an extensively broken C-N glycosidic bond, formation of an oxocarbenium ion in the ribose ring, C3′- exo ribose ring conformation, and almost no bond formation to the attacking nucleophile. The proposed transition state for the prokaryotic E. coli nucleoside hydrolase is compared to that of a similar enzyme isolated from Crithidia fasciculata ( C. fasciculata) .
Keywords: Abbreviations; LC/MS; liquid chromatography/mass spectrometry; IU–NH; inosine/uridine nucleoside hydrolase; GI–NH; guanosine/inosine preferring nucleoside hydrolase; KIE; kinetic isotope effects; LB; Luria–Bertani; IPTG; isopropyl β-; d; -thiogalactopyranoside; Tris; tris(hydroxymethyl)aminomethane; ODS; octadecyl; API-ES; atmospheric pressure interface-electrospray mass spectrometer; BEBOVIB; bond-energy bond-order vibrational analysis; SIM; selective ion monitoring; GC/MS; gas chromatography/mass spectrometry; SDS-PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresisKinetic isotope effect; Nucleoside hydrolase; Transition state
Comparison of functional properties of mammalian DNA polymerase λ and DNA polymerase β in reactions of DNA synthesis related to DNA repair by Natalia A. Lebedeva; Nadejda I. Rechkunova; Sergey V. Dezhurov; Svetlana N. Khodyreva; Alain Favre; Luis Blanco; Olga I. Lavrik (pp. 150-158).
DNA polymerase λ (Pol λ) is a novel enzyme of the family X of DNA polymerases. Pol λ has some properties in common with DNA polymerase β (Pol β). The substrate properties of Pol λ were compared to Pol β using DNAs mimicking short-patch (SP) and long-patch (LP) base excision repair (BER) intermediates as well as recessed template primers. In the present work, the influence of several BER proteins such as flap-endonuclease-1 (FEN1), PCNA, and apurinic/apyrimidinic endonuclease-1 (APE1) on the activity of Pol λ was investigated. Pol λ is unable to catalyze strand displacement synthesis using nicked DNA, although this enzyme efficiently incorporates a dNMP into a one-nucleotide gap. FEN1 and PCNA stimulate the strand displacement activity of Pol λ. FEN1 processes nicked DNA, thus removing a barrier to Pol λ DNA synthesis. It results in a one-nucleotide gapped DNA molecule that is a favorite substrate of Pol λ. Photocrosslinking and functional assay show that Pol λ is less efficient than Pol β in binding to nicked DNA. APE1 has no influence on the strand displacement activity of Pol λ though it stimulates strand displacement synthesis catalyzed with Pol β. It is suggested that Pol λ plays a role in the SP BER rather than contributes to the LP BER pathway.
Keywords: DNA polymerase λ; DNA polymerase β; Flap-endonuclease-1; Apurinic/apyrimidinic endonuclease-1; Base excision repair
Characterization of a dimeric unfolding intermediate of bovine serum albumin under mildly acidic condition by Amrita Brahma; Chhabinath Mandal; Debasish Bhattacharyya (pp. 159-169).
Protein aggregation is a well-known phenomenon related to serious medical implications. Bovine serum albumin (BSA), a structural analogue of human serum albumin, has a natural tendency for aggregation under stress conditions. While following effect of moderately acidic pH on BSA, a state was identified at pH 4.2 having increased light scattering capability at 350 nm. It was essentially a dimer devoid of disulphide linked large aggregates as observed from ‘spin column’ experiments, gel electrophoresis and ultra-centrifugations. Its surface hydrophobic character was comparable to the native conformer at pH 7.0 as observed by the extraneous fluorescence probes pyrene and pyrene maleimide but its interactions with 1-anilino 8-naphthelene sulphonic acid was more favorable. Dimerization was irreversible between pH 4.2 and 7.0 even after treatment with DTT. The role of the only cysteine-34 residue was investigated where modification with reagents of arm length bigger than 6 Å prevented dimerization. Molecular modeling of BSA indicated that cys-34 resides in a cleft of 6 Å depth. This indicated that the area surrounding the cleft plays important role in inducing the dimerization.
Keywords: Abbreviations; 1-ANS; 1-anilino 8-naphthylene sulfonic acid; BAL; sodium arsenite; BSA; Bovine serum albumin; CD; Circular dichroism; DTNB; dithio bis-trinitrobenzoic acid; NBD-Cl; 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole; NEM; N; -ethyl maleimide; MMTS; methyl methane thiosulphonate; PM; pyrene maleimideBSA; pH denaturation; Conformation; Aggregation; Cysteine-34
Mutagenesis of Ala290, which modulates substrate subsite affinity at the catalytic interface of dimeric ThMA by Sung-Hoon Park; Hyunju Cha; Hee-Kwon Kang; Jae-Hoon Shim; Eui-Jeon Woo; Jung-Wan Kim; Kwan-Hwa Park (pp. 170-177).
The goal of this study was to develop a maltose-producing enzyme using protein engineering and to clarify the relation between the substrate specificity and the structure of the substrate-binding site of dimeric maltogenic amylase isolated from Thermus (ThMA). Ala290 at the interface of ThMA dimer in the vicinity of the substrate-binding site was substituted with isoleucine, which may cause a structural change due to its bulky side chain. TLC analysis of the action pattern of the mutant ThMA-A290I, using maltooligosaccharides as substrates, revealed that ThMA-A290I used maltotetraose to produce mostly maltose, while wild-type ThMA produced glucose as well as maltose. The wild-type enzyme eventually hydrolyzed the maltose produced from maltotetraose into glucose, but the mutant enzyme did not. For both enzymes, the cleavage frequency of the glycosidic bond of maltooligosaccharides was the highest at the second bond from the reducing end. The mutant ThMA had a much higher Km value for maltose than the wild-type ThMA. The kinetic parameter, kcat/ Km, of ThMA-A290I for maltose was 48 times less than that of wild-type ThMA, suggesting that the subsite affinity and hydrolysis mode of ThMA were modulated by the residue located at the interface of ThMA dimer near the active site. The conformational rearrangement in the catalytic interface probably led to the change in the substrate binding affinity of the mutant ThMA. Our results provide basic information for the enzymatic preparation of high-maltose syrup.
Keywords: Kinetic parameter; Maltogenic amylase (ThMA); Maltose; Thermus; sp.
Transition state stabilization by six arginines clustered in the active site of creatine kinase by Michael J. Jourden; Paul R. Geiss; Michael J. Thomenius; Lindsay A. Horst; Melissa M. Barty; Melissa J. Brym; Guy B. Mulligan; Ryan M. Almeida; Betsy A. Kersteen; Nichole R. Myers; Mark J. Snider; Charles L. Borders Jr.; Paul L. Edmiston (pp. 178-183).
Six fully conserved arginine residues (R129, R131, R235, R291, R319, and R340) closely grouped in the nucleotide binding site of rabbit muscle creatine kinase (rmCK) were mutated; four to alanine and all six to lysine. Kinetic analyses in the direction of phosphocreatine formation showed that all four alanine mutants led to substantial losses of activity with three (R129A, R131A, and R235A ) having no detectable activity. All six lysine mutants retained variable degrees of reduced enzymatic activity. Static quenching of intrinsic tryptophan fluorescence was used to measure the binding constants for MgADP and MgATP. Nucleotide binding was at most only modestly affected by mutation of the arginine residues. Thus, the cluster of arginines seem to be primarily responsible for transition state stabilization which is further supported by the observation that none of the inactive mutants demonstrated the ability to form a transition analogue complex of MgADP·nitrate·creatine as determined by fluorescence quenching assays. As a whole, the results suggest that the most important role these residues play is to properly align the substrates for stabilization of the phosphoryl transfer reaction.
Keywords: Creatine kinase; Active site; Mutagenesis; MgATP binding; Fluorescence quenching
Over-expression, purification and characterization of the oligomerization dynamics of an invertebrate mitochondrial creatine kinase by Gregg G. Hoffman; W. Ross Ellington (pp. 184-193).
Mitochondrial creatine kinase (MtCK) plays a central role in energy homeostasis within cells that display high and variable rates of ATP turnover. Vertebrate MtCKs exist primarily as octamers but readily dissociate into constituent dimers under a variety of circumstances. MtCK is an ancient protein that is also found in invertebrates including sponges, the most primitive of all multi-cellular animals. We have cloned, expressed, and purified one of these invertebrate MtCKs from a marine polychaete worm, Chaetopterus variopedatus (CVMtCK). Size exclusion chromatography and dynamic light scattering (DLS) were used to characterize oligomeric state in comparison with that of octameric chicken sarcomeric isoform (SarMtCK). At protein concentrations >1 mg/ml, CVMtCK was predominantly octameric (>90%). When diluted to 0.1 mg/ml, CVMtCK dissociated into dimers much more rapidly than SarMtCK when observed under identical conditions. The rate of dissociation for both MtCKs increased as temperature rose from 2 to 28 °C, and in CVMtCK, fell at higher incubation temperatures. The fraction of octameric CVMtCK at equilibrium increased with temperature and then fell. Temperature transition studies showed that octamers and dimers were rapidly interconvertible on a similar time scale. Importantly, when CVMtCK was converted to the transition state analog complex (TSAC), both size exclusion chromatography and DLS showed that there was minimal dissociation of octamers into dimers while SarMtCK octamers were highly unstable as the TSAC. These results clearly show distinct differences in octamer stability between CVMtCK and SarMtCK, which could impact function under physiological circumstances. Furthermore, the large yield of recombinant protein and high stability of CVMtCK in the TSAC suggest that this protein might be a good target for crystallization efforts.
Keywords: Abbreviations; ANT; adenine nucleotide translocase; CM; carboxymethyl; DLS; dynamic light scattering; DTT; dithiothreitol; IMS; intermembrane space; IPTG; isopropyl-β-; d; -thiogalactopyranoside; β-MSH; β-mercaptoethanol; PCr; phosphocreatine; MtCK; mitochondrial creatine kinase; CVMtCK; Chaetopterus variopedatus; MtCK; ROS; reactive oxygen species; SarMtCK; sarcomeric MtCK; TSAC; transition state analog complex; UMtCK; ubiquitous MtCKCreatine kinase; Oligomeric state; Transition state analog complex; Mitochondria
MnlI—The member of H-N-H subtype of Type IIS restriction endonucleases by Edita Kriukiene; Judita Lubiene; Arunas Lagunavicius; Arvydas Lubys (pp. 194-204).
The Type IIS restriction endonuclease MnlI recognizes the non-palindromic nucleotide sequence 5′-CCTC(N)7/6↓ and cleaves DNA strands as indicated by the arrow. The genes encoding MnlI restriction–modification system were cloned and sequenced. It comprises N6-methyladenine and C5-methylcytosine methyltransferases and the restriction endonuclease. Biochemical studies revealed that MnlI restriction endonuclease cleaves double- and single-stranded DNA, and that it prefers different metal ions for hydrolysis of these substrates. Mg2+ ions were shown to be required for the specific cleavage of double-stranded DNA, whereas Ni2+ and some other transition metal ions were preferred for nonspecific cleavage of single-stranded DNA. The C-terminal part of MnlI restriction endonuclease revealed an intriguing similarity with the H-N-H type nucleolytic domain of bacterial toxins, Colicin E7 and Colicin E9. Alanine replacements in the conserved sequence motif 306Rx3ExHHx14Nx8H greatly reduced specific activity of MnlI, and some mutations even completely inactivated the enzyme. However, none of these mutations had effect on MnlI binding to the specific DNA, and on its oligomerisation state as well. We interpret the presented experimental evidence as a suggestion that the motif 306Rx3ExHHx14Nx8H represents the active site of MnlI. Consequentially, MnlI seems to be the member of Type IIS with the active site of the H-N-H type.
Keywords: Restriction–modification system; Restriction endonuclease; REase; MnlI; H-N-H motif; Colicin
Crystallization and preliminary X-ray crystallographic analysis of the pediocin immunity protein (PedB) from Pediococcus pentosaceus at 1.35 Å resolution by In-Kwon Kim; Min-Kyu Kim; Hyung-Soon Yim; Sun-Shin Cha; Sa-Ouk Kang (pp. 205-208).
PedB, a bacterial immunity protein conferring immunity to a newly identified pediocin (pediocin PP-1), was crystallized by the hanging-drop vapor diffusion method at 296 K. A 1.35 Å data set has been collected from a single crystal at 100 K using synchrotron-radiation source. The PedB crystals belong to the hexagonal space group P62 or P64, with unit cell parameters a= b=62.2, c=39.9 Å. Analysis of the packing density shows that the asymmetric unit probably contains one molecule with a solvent content of 33.8%.
Keywords: PedB; Bacteriocin; Pediocin; Immunity protein
Crystallization and preliminary crystallographic studies of the recombinant antitumour lectin from the edible mushroom Agrocybe aegerita by Na Yang; Xin Tong; Ye Xiang; Ying Zhang; Hui Sun; Da-Cheng Wang (pp. 209-212).
The antitumour lectin from Agrocybe aegerita, named AAL, shows strong inhibition effects on human and mouse tumour cells via apoptosis induction activity. Recombinant AAL (rAAL) has been expressed and purified. Both rAAL and rAAL–lactose complex have been crystallized and their X-ray diffraction data were collected to resolutions of 1.9 Å and 1.6 Å, respectively. Both crystals belong to space group P21 with unit cell parameters a=53.20 Å, b=66.01 Å, c=57.86 Å, β=109.38 and a=53.38 Å, b=66.29 Å, c=58.02 Å, β=109.03, respectively.
Keywords: Lectin; Mushroom; Antitumour; Apoptosis; Crystallization
Crystallization of bacteriorhodopsin solubilized by a tripod amphiphile by Michael J. Theisen; Terra B. Potocky; D. Tyler McQuade; Samuel H. Gellman; Mark L. Chiu (pp. 213-216).
Bacteriorhodopsin (bR) is solubilized efficiently as a monomer by a novel surfactant, a tripod amphiphile (TPA), which permits the formation of purple hexagonal bR crystals under several conditions. The crystals, although small, diffract to 2.5 Å resolution using synchrotron radiation. TPA may be useful for the solubilization, purification, and crystallization of other membrane proteins.
Keywords: Membrane protein; Crystallization; Bacteriorhodopsin; Tripod amphiphile; Detergent; Surfactant