Young Kee Chae

Structure-function studies of [2Fe-2S] ferredoxins

The ability to overexpress [2Fe-2S] ferredoxins inEscherichia coli has opened up exciting research opportunities. High-resolution x-ray structures have been determined for the wild-type ferredoxins produced by the vegetative and heterocyst forms ofAnabaena strain 7120 (in their oxidized states), and these have been compared to structural information derived from multidimensional, multinuclear NMR spectroscopy. The electron delocalization in these proteins in their oxidized and reduced states has been studied by1H,2H,13C, and15N NMR spectroscopy. Site-directed mutagenesis has been used to prepare variants of these ferredoxins. Mutants (over 50) of the vegetative ferredoxin have been designed to explore questions about cluster assembly and stabilization and to determine which residues are important for recognition and electron transfer to the redox partnerAnabaena ferredoxin reductase. The results have shown that serine can replace cysteine at each of the four cluster attachment sites and still support cluster assembly. Electron transfer has been demonstrated with three of the four mutants. Although these mutants are less stable than the wild-type ferredoxin, it has been possible to determine the x-ray structure of one (C49S) and to characterize all four by EPR and NMR. Mutagenesis has identified residues 65 and 94 of the vegetative ferredoxin as crucial to interaction with the reductase. Three-dimensional models have been obtained by x-ray diffraction analysis for several additional mutants: T48S, A50V, E94K (four orders of magnitude less active than wild type in functional assays), and A43S/A45S/T48S/A50N (quadruple mutant).

Lipid Binding Ridge on Loops 2 and 3 of the C2A Domain of Synaptotagmin I as Revealed by NMR Spectroscopy

The C2A domain of synaptotagmin I, which binds Ca2+ and anionic phospholipids, serves as a Ca2+ sensor during excitation-secretion coupling. We have used multidimensional NMR to locate the region of C2A from rat synaptotagmin I that interacts, in the presence of Ca2+, with phosphatidylserine. Untagged, recombinant C2A was double-labeled with 13C and15N, and triple-resonance NMR data were collected from C2A samples containing either Ca2+ alone or Ca2+ plus 6:0 phosphatidylserine. Phospholipid binding led to changes in chemical shifts of backbone atoms in residues Arg233 and Phe234 of loop 3 (a loop that also binds Ca2+) and His198, Val205, and Phe206 of loop 2. These residues lie along a straight line on a surface ridge of the C2A domain. The only other residue that exhibited appreciable chemical shift changes upon adding lipid was His254; however, because His254 is located on the other side of the molecule from the phospholipid docking site defined by the other residues, its shifts may result from nonspecific interactions. The results show that the “docking ridge” responsible for Ca2+-dependent membrane association is localized on the opposite side of the C2A domain from the transmembrane and C2B domains of synaptotagmin.

Regulation Of In Vitro Fibril Formation Of Synuclein Mutant Proteins By Hsp104p

Hsp104p, as an anti-oxidative protector of ROS generation, was examined to inquire if it prevents aggregation of synuclein mutants, A30P or A53T upon aging, in vitro. The role of Hsp104p was also addressed in dissociation of pre-formed aggregates of synuclein mutants. Significant protection in fibril formation was observed by wild-type Hsp104p regardless of ATP presence, not by mutant Hsp104p. To a lesser extent, the dissociation effect of wild-type Hsp104p was observed only in the presence of ATP. These results will be discussed in relation to the development of an antioxidant approach to prevent amyloid fibril formation in several neurodegenerative diseases.

Crystallization and preliminary analysis of oxidized, recombinant, heterocyst [2Fe2S] ferredoxin from Anabaena 7120

The [2Fe-2S] ferredoxin produced in the heterocyst cells of Anabaena 7120 plays a key role in nitrogen fixation, where it serves as an electron acceptor from various sources and an electron donor to nitrogenase. Crystals of recombinant heterocyst ferredoxin, coded for by the fdx H gene from Anabaena 7120 and overproduced in Escherichia coli, have been grown from ammonium sulfate solutions and are suitable for high resolution X-ray crystallographic analysis. They belong to the hexagonal space group P6(1) or P6(5) with unit cell dimensions of a = b = 44.2 A and c = 80.6 A. The crystals contain one molecule per asymmetric unit and diffract to a nominal resolution of 1.6 A. The molecular structure of this heterocyst ferredoxin is of special interest in that 4 of the 22 amino acid positions thought to be absolutely conserved in nonhalophilic ferredoxins are different and, based on amino acid sequence alignments, three of these positions are located in the metal-cluster binding loop. Consequently, a high-resolution X-ray analysis of this [2Fe-2S] ferredoxin, and subsequent three-dimensional comparisons with other known ferredoxin models, will provide new insight into structure/function relationships for this class of redox proteins.

Stress adaptation of Saccharomyces cerevisiae as monitored via metabolites using two‐dimensional NMR spectroscopy

Many studies on yeast metabolism are focused on its response to specific stress conditions because the results can be extended to the human medical issues. Most of those works have been accomplished through functional genomics studies. However, these changes may not show a linear correlation with protein or metabolite levels. For many organisms including yeast, the number of metabolites is far fewer than that of genes or gene products. Thus, metabolic profiling can provide a simpler yet efficient snapshot of the systems physiology. Metabolites of Saccharomyces cerevisiae under various stresses were analyzed and compared with those under the normal, unstressed growth conditions by two-dimensional NMR spectroscopy. At least 31 metabolites were identified for most of the samples. The levels of many identified metabolites showed significant increase or decrease depending on the nature of the stress. The statistical analysis produced a holistic view: different stresses were clustered and isolated from one another with the exception of high pH, heat, and oxidative stresses. This work could provide a link between the metabolite profiles and mRNA or protein profiles under representative and well-studied stress conditions.

Protein Production By Stationary Phase Induction (Spi)

An alternative method for expressing the recombinant proteins in Escherichia coli is proposed. Unlike the ordinary induction protocol where the cells in the early- to mid-log phase are induced for the protein production, this alternative protocol utilizes the cells in the stationary phase. By using a glutathione S-transferase fusion protein as an example, the protocol proposed in this report yielded a higher amount of the desired protein than that from the ordinary protocol. This protocol also suppressed the proteolytic cleavage of the desired protein in the Escherichia coli cytoplasm, thus it should be particularly useful to produce proteins that undergo unwanted cleavages.

Application of two-dimensional NMR spectroscopy to metabotyping laboratory Escherichia coli strains.

NMR Spectroscopy has been established as a major tool for identification and quantification of metabolites in a living system. Since the metabolomics era began, one‐dimensional NMR spectroscopy has been intensively employed due to its simplicity and quickness. However, it has suffered from an inevitable overlap of signals, thus leading to inaccuracy in identification and quantification of metabolites. Two‐dimensional (2D) NMR has emerged as a viable alternative because it can offer higher accuracy in a reasonable amount of time. We employed 1H,13C‐HSQC to profile metabolites of six different laboratory E. coli strains. We identified 18 metabolites and observed clustering of six strains according to their metabolites. We compared the metabolites among the strains, and found that a) the strains specialized for protein production were segregated; b) XL1‐Blue separated itself from others by accumulating amino acids such as alanine, aspartate, glutamate, methionine, proline, and lysine; c) the strains specialized for cloning purpose were spread out from one another; and d) the strains originating from B strain were characterized by succinate accumulation. This work shows that 2D‐NMR can be applied to identify a strain from metabolite analysis, offering a possible alternative to genetic analysis to identify E. coli strains.

The Native Metastability and Misfolding of Serine Protease Inhibitors

The native metastability of serine protease inhibitors (serpins) is believed to facilitate the conformational change required for biological function. However, energetically unfavorable structural features that contribute to metastability of the native serpin conformation, such as buried polar groups, cavities, and over-packing of side-chains, also appear to hinder proper folding. Hence, folding of serpin polypeptides appears prone to error; in particular, the folding polypeptides are readily diverted toward a non-productive folding pathway culminating in a more stable but inactive conformation. In a survey of deficient serpin mutants, various folding defects, such as retarded protein folding, destabilized native conformation, and spontaneous conversion into more stable, inactive conformations such as the latent form and loop-sheet polymers, have been discovered.

A Prionogenic Peptide Derived from Sup35 can Force the Whole Gst Fusion Protein to Show Amyloid Characteristics

A prion determining 7-mer peptide derived from Sup35 was fused to glutathione S transferase (GST). The fusion protein was successfully overexpressed in Escherichia coli, and purified by employing affinity chromatography. Upon incubation, it showed substantial aggregation suggesting the formation of amyloid-like fibrils. Congo Red binding strongly suggested that the fusion protein formed amyloid-like fibrils. By considering the steric hindrance of GST, the beta-sheet formation should be in the anti-parallel fashion.

Probing Metabolite Space of Escherichia coli via Growth Medium Composition as Monitored by Two-Dimensional NMR Spectroscopy.

As for recombinant protein production, Escherichia coli is one of the most frequently employed hosts because it offers a simple and inexpensive, but rapid and high-yield system in addition to the vast information on its molecular genetics and biology. However, due to its prokaryotic nature, it often fails to produce eukaryotic proteins in a desired form. To devise a systematic way leading to a condition that produces a large amount of usable proteins, we attempted to monitor intracellular metabolites under various conditions, and to link them to recombinant protein production. With such an intention, we identified 31 metabolites from cells grown in different media by using two-dimensional (2D) NMR spectroscopy. Our results revealed that 1) the level of betaine was low, while that of glutamic acid was high when grown in minimal media; 2) the level of glycerol was constantly high in all cases; 3) the level of oxidized glutathione was lower in Luria broth (LB); and 4) the level of leucine was low in minimal media. We hope this work might shed light onto how to improve production of the target proteins by metabolite profiling.As for recombinant protein production, Escherichia coli is one of the most frequently employed hosts because it offers a simple and inexpensive, but rapid and high‐yield system in addition to the vast information on its molecular genetics and biology. However, due to its prokaryotic nature, it often fails to produce eukaryotic proteins in a desired form. To devise a systematic way leading to a condition that produces a large amount of usable proteins, we attempted to monitor intracellular metabolites under various conditions, and to link them to recombinant protein production. With such an intention, we identified 31 metabolites from cells grown in different media by using two‐dimensional (2D) NMR spectroscopy. Our results revealed that 1) the level of betaine was low, while that of glutamic acid was high when grown in minimal media; 2) the level of glycerol was constantly high in all cases; 3) the level of oxidized glutathione was lower in Luria broth (LB); and 4) the level of leucine was low in minimal media. We hope this work might shed light onto how to improve production of the target proteins by metabolite profiling.