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PubMed2005

A1 reduction in intact cyanobacterial photosystem I particles studied by time-resolved step-scan Fourier transform infrared difference spectroscopy and isotope labeling.

Time-resolved step-scan Fourier transform infrared (FTIR) difference spectroscopy, with 5 mus time resolution, has been used to produce P700(+)A(1)(-)/P700A(1) FTIR difference spectra in intact photosystem I particles from Synechococcus sp. 7002 and Synechocystis sp. 6803 at 77 K. Corresponding spectra were also obtained for fully deuterated photosystem I particles from Synechococcus sp. 7002 as well as fully (15)N- and (13)C-labeled photosystem I particles from Synechocystis sp. 6803. Static P700(+)/P700 FTIR difference spectra at 77 K were also obtained for all of the unlabeled and labeled photosystem I particles. From the time-resolved and static FTIR difference spectra, A(1)(-)/A(1) FTIR difference spectra were constructed. The A(1)(-)/A(1) FTIR difference spectra obtained for unlabeled trimeric photosystem I particles from both cyanobacterial strains are very similar. There are some mode frequency differences in spectra obtained for monomeric and trimeric PS I particles. However, the spectra can be interpreted in an identical manner, with the proposed band assignments being compatible with all of the data obtained for labeled and unlabeled photosystem I particles. In A(1)(-)/A(1) FTIR difference spectra obtained for unlabeled photosystem I particles, negative bands are observed at 1559 and 1549-1546 cm(-)(1). These bands are assigned to amide II protein vibrations, as they downshift approximately 86 cm(-)(1) upon deuteration and approximately 13 cm(-)(1) upon (15)N labeling. Difference band features at 1674-1677(+) and 1666(-) cm(-)(1) display isotope-induced shifts that are consistent with these bands being due to amide I protein vibrations. The observed amide modes suggest alteration of the protein backbone (possibly in the vicinity of A(1)) upon A(1) reduction. A difference band at 1754(+)/1748(-) cm(-)(1) is observed in unlabeled spectra from both strains. The frequency of this difference band, as well as the observed isotope-induced shifts, indicate that this difference band is due to a 13(3) ester carbonyl group of chlorophyll a species, most likely the A(0) chlorophyll a molecule that is in close proximity to A(1). Thus A(1) reduction perturbs A(0), probably via a long-range electrostatic interaction. A negative band is observed at 1693 cm(-)(1). The isotope shifts associated with this band are consistent with this band being due to the 13(1) keto carbonyl group of chlorophyll a, again, most likely the 13(1) keto carbonyl group of the A(0) chlorophyll a that is close to A(1). Semiquinone anion bands are resolved at approximately 1495(+) and approximately 1414(+) cm(-)(1) in the A(1)(-)/A(1) FTIR difference spectra for photosystem I particles from both cyanobacterial strains. The isotope-induced shifts of these bands could suggest that the 1495(+) and 1414(+) cm(-)(1) bands are due to C-O and C-C modes of A(1)(-), respectively.

Amides↗
PubMed2006

Recombinant expression, isotope labeling, refolding, and purification of an antimicrobial peptide, piscidin.

An antimicrobial peptide, piscidin, was overexpressed as a fused form with the ubiquitin molecule in Escherichia coli, and the fusion protein was purified using immobilized metal affinity chromatography (IMAC). The peptide was released from its fusion partner by using yeast ubiquitin hydrolase (YUH), and subsequently purified by reverse phase chromatography. The expression and purification process of piscidin encountered several problems such as the lysis of the bacterial cell upon induction of the peptide production, the unwanted cleavage of the fusion protein inside the bacterial cell, and high tendency to aggregate in the aqueous environment. Such problems were alleviated by employing ubiquitin as a fusion partner for piscidin, growing the cells at a lower temperature, and changing the order of the purification steps. The yields of the fusion protein and the peptide were around 15 and 1.5 mg per liter of LB or minimal medium, respectively. The recombinant expression and purification of piscidin will enable its structural and dynamic studies using multidimensional NMR spectroscopy.

Animals↗
PubMed2005

Expression and isotopic labeling of structural domains of the human protein DEK.

The 375 amino acid human protein DEK has been expressed in two functional, structured domains. DEK is an abundant nuclear protein that associates with chromatin and alters its topology by introducing positive supercoiling in DNA, which results in lower replication efficiency. DEK has clinical importance as transfection of the cDNA of the C-terminal region of DEK can partially reverse the abnormal DNA-mutagen sensitivity in fibroblasts derived from ataxia-telangiectasia (A-T) patients, and elevated levels of DEK mRNA are observed in various forms of cancer. Because high-level expression of full-length DEK has proved elusive, we sought an alternative for structural studies that would provide insights on DEK's function. We have discovered that DEK contains two structured domains and have expressed these domains at a high level in Escherichia coli in M9 minimal media. The N-terminal domain (amino acids 68-226) includes the region responsible for introducing supercoils into DNA, and the C-terminal domain (amino acids 309-375) includes the region that can reverse the abnormal DNA-mutagen sensitivity of A-T cells. 1H-15N correlation nuclear magnetic resonance spectra of these two fragments reveal the characteristic signature of folded proteins.

Chromosomal Proteins, Non-Histone↗
PubMed2005

Preparation of isotopically labeled spinach acyl-acyl carrier protein for NMR structural studies.

Acyl carrier proteins (ACPs) are important protein cofactors in fatty acid biosynthesis, but their acylated forms have not been well-studied. To permit detailed nuclear magnetic resonance studies of acylated spinach ACP isoform I, we have developed a new expression plasmid for recombinant production of the apo-protein and modified protocols for purifying the protein product and acylating it to form acyl-ACP. To solve plasmid stability problems associated with growth in minimal media, the ampicillin resistance gene from pSACP-2a was replaced with the tetA(C) gene from pBR322. The resulting plasmid, pSACP-2t, supported overexpression of apo-ACP in Escherichia coli BL21(DE3) cells in M9 medium containing 15NH4Cl as the sole nitrogen source. Apo-ACP was purified to homogeneity by means of polyethylene glycol precipitation and anion exchange. Two in vitro synthetic routes were used to produce acyl-ACPs. In one route, apo-ACP was converted to the holo form and the acyl form by a published protocol that employs a discrete enzymatic reaction for each step. As an alternative route to produce decanoyl-ACP, apo-ACP was directly converted to the acyl form by using holo-ACP synthase along with the non-natural substrate decanoyl-CoA. Two-dimensional 1H-15N NMR spectroscopy of decanoyl-ACP and stearoyl-ACP revealed that changes in the length of the covalently attached fatty acid do not affect the secondary structure of the protein but do influence the local conformation and dynamics.

Acyl Carrier Protein↗
PubMed2003

Stable isotope labeling of a Group A Streptococcus virulence factor using a chemically defined growth medium.

A secreted, hypervariable virulence factor called the streptococcal inhibitor of complement (Sic) has been linked to the reemergence of epidemics due to the human pathogenic bacterium Group A Streptococcus. This paper describes a method for expressing and purifying Sic from an attenuated GAS strain using a chemically defined growth medium. This method was used to label specific amino acid residue types in Sic with forms containing the magnetically active isotope (15)N, at the amide nitrogen. The (15)N-labeling of Sic permits a detailed investigation of the structure and dynamics of the protein using nuclear magnetic resonance spectroscopy. The level of stable isotope incorporation was established using mass spectrometry and nuclear magnetic resonance spectroscopy.

Amides↗
PubMed1992

Stable isotope labeling of lead compartments in rats with ultralow lead concentrations.

The role of the mammalian skeleton as an endogenous lead source is unclear. This is due in part to difficulties in distinguishing mobilized skeletal lead from other endogenous and exogenous lead sources. Therefore, we have applied ultraclean stable lead isotope techniques to label skeletal and soft tissue lead compartments within the rat with distinguishable lead isotopic signatures. Female Wistar (defined flora) rats were fed 206Pb-enriched drinking water ([Pb] = 110 ng/ml) and sacrificed after durations of 2, 4, 7, and 14 days. Blood, kidney, vertebra, and tibia tissues were analyzed for lead concentrations and stable isotopic compositions. The resulting isotopic ratios in soft (blood and kidney) and skeletal (vertebrae and tibia) tissues differed by approximately 40% after 2 days exposure to the 206Pb tracer. More than 90% of the tracer isotopic signature was contained in the soft tissues after 10 days exposure, while skeletal tissues acquired only approximately 50% of the tracer by the end of the study. Because these animals were maintained under trace metal-clean conditions, they contained lead concentrations in whole blood (0.3-3 ng/g), kidney (11-27 ng/g dry wt), and bone (35-70 ng/g dry wt) tissues that are the lowest known reported for contemporary terrestrial mammals, and they (in bone) are comparable to levels in preindustrial mammals. The elevated concentrations of lead in kidney (fresh weight) relative to levels in blood are consistent with the presence of specific lead-binding sites in the kidney at very low levels of exposure.

Animals↗