Recombinant PAS-heme domains of oxygen sensing proteins: High level production and physical characterization

Suquet, Christine; Savenkova, Marina I.; Satterlee, James D.

doi:10.1016/j.pep.2005.03.002

Cited by 14 publications

(37 citation statements)

References 34 publications

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“…The gene was provided by Dr. J. D. Satterlee. Protein overexpression and purification were carried out following published protocols (Suquet et al, 2005) with minor modifications. The apoprotein was obtained from inclusion bodies, and the holoprotein was prepared by reconstitution with hemin.…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

Structural and thermodynamic consequences of b heme binding for monomeric apoglobins and other apoproteins

Landfried

Vuletich

Pond

et al. 2007

Gene

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The binding of a cofactor to a protein matrix often involves a reorganization of the polypeptide structure. b Hemoproteins provide multiple examples of this behavior. In this minireview, selected monomeric and single b heme proteins endowed with distinct topological properties are inspected for the extent of induced refolding upon heme binding. To complement the data reported in the literature, original results are presented on a two-on-two globin of cyanobacterial origin (Synechococcus sp. PCC 7002 GlbN) and on the heme-containing module of FixL, an oxygen-sensing protein with the mixed alpha/beta topology of PAS domains. GlbN had a stable apoprotein that was further stabilized and locally refolded by heme binding; in contrast, apoFixLH presented features of a molten globule. Sequence analyses (helicity, disorder, and polarity) and solvent accessibility calculations were performed to identify trends in the architecture of b hemoproteins. In several cases, the primary structure appeared biased toward a partially disordered binding pocket in the absence of the cofactor.

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Section: Protein Expression and Purificationmentioning

confidence: 99%

Structural and thermodynamic consequences of b heme binding for monomeric apoglobins and other apoproteins

Landfried

Vuletich

Pond

et al. 2007

Gene

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“…The production techniques, including various primers, isolation and purification procedures and all chemicals and procedures for the recombinant native and variant BjFixLH proteins used in this work have been described in detail previously [1,28]. Primary sequencing of the amino termini was carried out as described previously [28].…”

Section: General Methodsmentioning

confidence: 99%

“…Primary sequencing of the amino termini was carried out as described previously [28]. Initially purified preparations of the expressed parent protein of the three variants described herein, BjFixLH 140-270 , are mass homogeneous as indicated by MALDI-TOF (Matrix Assisted Laser Desorption Ionization Time of Flight) mass spectrometry [1,28]. All solutions were made using 18 MΩ water (Barnstead) that was also filter sterilized.…”

Section: General Methodsmentioning

confidence: 99%

“…All solutions were made using 18 MΩ water (Barnstead) that was also filter sterilized. Purity and homogeneity assessments of each protein preparation by mass spectrometry were carried out as previously described [1,28]. Pertinent to the question of the origin of the observed mass losses in all the recombinant BjFixL heme domains from this laboratory is the fact that the isolation and purification procedures always included addition of protease inhibitor cocktail (Sigma P2714; P8465) and separately AEBSF (Sigma A8456) (early work was with PMSF) when the E. coli BL21 cells were lysed at the start of isolation.…”

Section: General Methodsmentioning

confidence: 99%

“…Purified solutions were filter sterilized, refreshed with protease inhibitors and stored at −80°C. Details of MALDI-TOF mass spectrometry carried out on a Voyager DE system (Perseptive Biosystems) in linear mode were identical to those previously described [1,28], including the matrix (sinapic acid, Sigma). Mass calibrations utilized 3-point (equine cytochrome c +1 ion and equine myoglobin +1 and +2 ion) and 2-point (myoglobin only) internal references.…”

Section: General Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Origins of aging mass loss in recombinant N-terminus and C-terminus deletion mutants of the heme-PAS biosensor domain BjFixLH140–270

Satterlee

2011

Journal of Inorganic Biochemistry

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Keeping PASE with WEFT: SHWEFT–PASE pulse sequences for ¹H NMR spectra of highly paramagnetic molecules

Helms

Satterlee

2013

Magnetic Reson in Chemistry

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Metalloproteins are a category of biomolecules in which the metal site is usually the locus of activity or function. In many cases, the metal ions are paramagnetic or have accessible paramagnetic states, many of which can be studied using NMR spectroscopy. Extracting useful information from (1)H NMR spectra of highly paramagnetic proteins can be difficult because the paramagnetism leads to large resonance shifts (~400 ppm), extremely broad lines, extreme baseline nonlinearity, and peak shape distortion. It is demonstrated that employing polychromatic and adiabatic shaped pulses in simple pulse sequences, then combining existing sequences, leads to significant spectral improvement for highly paramagnetic proteins. These sequences employ existing technology, with available hardware, and are of short duration to accommodate short nuclear T1 and T2. They are shown to display uniform excitation over large spectral widths (~75 kHz), accommodate high repetition rates, produce flat baselines over 75 kHz while maintaining peak shape fidelity, and can be used to reduce spectral dynamic range. High-spin (S = 5/2) metmyoglobin, a prototypical highly paramagnetic protein, was used as the test molecule. The resulting one-dimensional (1D) pulse sequences combine shaped pulses with super-water elimination Fourier transform, which can be further combined with paramagnetic spectroscopy to give shaped pulses with super-water elimination Fourier transform-paramagnetic spectroscopy. These sequences require, at most, direct current offset correction and minimal phasing. The performance of these sequences in simple (1)H 1D, 1D NOE, and two-dimensional NOESY experiments is demonstrated for metmyoglobin and Paracoccus denitrificans Co(2+)-amicyanin (S = 3/2), and employed to make new heme hyperfine resonance assignments for high-spin metBjFixLH(151-256), the heme sensing domain of Bradyrhizobium japonicum FixL.

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Recombinant PAS-heme domains of oxygen sensing proteins: High level production and physical characterization

Cited by 14 publications

References 34 publications

Structural and thermodynamic consequences of b heme binding for monomeric apoglobins and other apoproteins

Structural and thermodynamic consequences of b heme binding for monomeric apoglobins and other apoproteins

Origins of aging mass loss in recombinant N-terminus and C-terminus deletion mutants of the heme-PAS biosensor domain BjFixLH140–270

Keeping PASE with WEFT: SHWEFT–PASE pulse sequences for ¹H NMR spectra of highly paramagnetic molecules

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Recombinant PAS-heme domains of oxygen sensing proteins: High level production and physical characterization

Cited by 14 publications

References 34 publications

Structural and thermodynamic consequences of b heme binding for monomeric apoglobins and other apoproteins

Structural and thermodynamic consequences of b heme binding for monomeric apoglobins and other apoproteins

Origins of aging mass loss in recombinant N-terminus and C-terminus deletion mutants of the heme-PAS biosensor domain BjFixLH140–270

Keeping PASE with WEFT: SHWEFT–PASE pulse sequences for 1H NMR spectra of highly paramagnetic molecules

Contact Info

Product

Resources

About

Keeping PASE with WEFT: SHWEFT–PASE pulse sequences for ¹H NMR spectra of highly paramagnetic molecules