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ABSTRACT: The ubiquitous mitochondrial J-protein Jac1, called HscB in Escherichia coli, and its partner Hsp70 play a critical role in the transfer of Fe-S clusters from the scaffold protein Isu to recipient proteins. Biochemical results from eukaryotic and prokaryotic systems indicate that formation of the Jac1-Isu complex is important for both targeting of the Isu for Hsp70 binding and stimulation of Hsp70's ATPase activity. However, in apparent contradiction, we previously reported that an 8-fold decrease in Jac1's affinity for Isu1 is well tolerated in vivo, raising the question as to whether the Jac1:Isu interaction actually plays an important biological role. Here, we report the determination of the structure of Jac1 from Saccharomyces cerevisiae. Taking advantage of this information and recently published data from the homologous bacterial system, we determined that a total of eight surface-exposed residues play a role in Isu binding, as assessed by a set of biochemical assays. A variant having alanines substituted for these eight residues was unable to support growth of a jac1-Δ strain. However, replacement of three residues caused partial loss of function, resulting in a significant decrease in the Jac1:Isu1 interaction, a slow growth phenotype, and a reduction in the activity of Fe-S cluster-containing enzymes. Thus, we conclude that the Jac1:Isu1 interaction plays an indispensable role in the essential process of mitochondrial Fe-S cluster biogenesis.
Journal of Molecular Biology 03/2012; 417(1-2):1-12. · 4.00 Impact Factor
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Youngchang Kim,
Pearl Quartey,
Hui Li,
Lour Volkart,
Catherine Hatzos,
Changsoo Chang,
Boguslaw Nocek,
Marianne Cuff,
Jerzy Osipiuk,
Kemin Tan,
Yao Fan, Lance Bigelow,
Natalia Maltseva,
Ruiying Wu,
Maria Borovilos,
Erika Duggan,
Min Zhou,
T Andrew Binkowski,
Rong-guang Zhang,
Andrzej Joachimiak
Nature Methods 11/2008; 5(10):853-4. · 19.28 Impact Factor
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Youngchang Kim, Lance Bigelow,
Maria Borovilos,
Irina Dementieva,
Erika Duggan,
William Eschenfeldt,
Catherine Hatzos,
Grazyna Joachimiak,
Hui Li,
Natalia Maltseva,
Rory Mulligan,
Pearl Quartey,
Alicia Sather,
Lucy Stols,
Lour Volkart,
Ruiying Wu,
Min Zhou,
Andrzej Joachimiak
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ABSTRACT: In structural biology, the most critical issue is the availability of high-quality samples. "Structural-biology-grade" proteins must be generated in a quantity and quality suitable for structure determination using X-ray crystallography or nuclear magnetic resonance. The additional challenge for structural genomics is the need for high numbers of proteins at low cost where protein targets quite often have low sequence similarities, unknown properties and are poorly characterized. The purification procedures must reproducibly yield homogeneous proteins or their derivatives containing marker atom(s) in milligram quantities. The choice of protein purification and handling procedures plays a critical role in obtaining high-quality protein samples. Where the ultimate goal of structural biology is the same-to understand the structural basis of proteins in cellular processes, the structural genomics approach is different in that the functional aspects of individual protein or family are not ignored, however, emphasis here is on the number of unique structures, covering most of the protein folding space and developing new technologies with high efficiency. At the Midwest Center Structural Genomics (MCSG), we have developed semiautomated protocols for high-throughput parallel protein purification. In brief, a protein, expressed as a fusion with a cleavable affinity tag, is purified in two immobilized metal affinity chromatography (IMAC) steps: (i) first IMAC coupled with buffer-exchange step, and after tag cleavage using TEV protease, (ii) second IMAC and buffer exchange to clean up cleaved tags and tagged TEV protease. Size exclusion chromatography is also applied as needed. These protocols have been implemented on multidimensional chromatography workstations AKTAexplorer and AKTAxpress (GE Healthcare). All methods and protocols used for purification, some developed in MCSG, others adopted and integrated into the MCSG purification pipeline and more recently the Center for Structural Genomics of Infectious Disease (CSGID) purification pipeline, are discussed in this chapter.
Advances in protein chemistry and structural biology. 01/2008; 75:85-105.
