Sotaro Fujii

High Thermal Stability and Unique Trimer Formation of Cytochrome c′ from Thermophilic Hydrogenophilus thermoluteolus

Sequence analysis indicated that thermophilic Hydrogenophilus thermoluteolus cytochrome c′ (PHCP) and its mesophilic homolog, Allochromatium vinosum cytochrome c′ (AVCP), closely resemble each other in a phylogenetic tree of the cytochrome c′ family, with 55% sequence identity. The denaturation temperature of PHCP was 87 °C, 35 °C higher than that of AVCP. Furthermore, PHCP exhibited a larger enthalpy change value during its thermal denaturation than AVCP. While AVCP was dimeric, as observed previously, PHCP was trimeric, and this was the first observation as a cytochrome c′. Dissociation of trimeric PHCP and its protein denaturation reversibly occurred at the same time in a two-state transition manner. Therefore, PHCP is enthalpically more stable than AVCP, perhaps due to its unique trimeric form, in addition to the lower number of Gly residues in its putative α-helical regions.

Comparative study on stabilization mechanism of monomeric cytochrome c5 from deep-sea piezophilic Shewanella violacea

Monomeric cytochrome c5 from deep-sea piezophilic Shewanella violacea (SVcytc5) was stable against heat and denaturant compared with the homologous protein from shallow-sea piezo-sensitive Shewanella livingstonensis (SLcytc5). Here, the SVcytc5 crystal structure revealed that the Lys-50 side chain on the flexible loop formed a hydrogen bond with heme whereas that of corresponding hydrophobic Leu-50 could not form such a bond in SLcytc5, which appeared to be one of possible factors responsible for the difference in stability between the two proteins. This structural insight was confirmed by a reciprocal mutagenesis study on the thermal stability of these two proteins. As SVcytc5 was isolated from a deep-sea piezophilic bacterium, the present comparative study indicates that adaptation of monomeric SVcytc5 to high pressure environments results in stabilization against heat. Graphical abstract Structure of Shewanella violaceacy to chrome c5 revealed that Lys-50 on a flexible loop was responsible for its stability.

Structural and functional insights into thermally stable cytochrome c' from a thermophile

Thermophilic Hydrogenophilus thermoluteolus cytochrome c′ (PHCP) exhibits higher thermal stability than a mesophilic counterpart, Allochromatium vinosum cytochrome c′ (AVCP), which has a homo‐dimeric structure and ligand‐binding ability. To understand the thermal stability mechanism and ligand‐binding ability of the thermally stable PHCP protein, the crystal structure of PHCP was first determined. It formed a homo‐dimeric structure, the main chain root mean square deviation (rmsd) value between PHCP and AVCP being 0.65 Å. In the PHCP structure, six specific residues appeared to strengthen the heme‐related and subunit–subunit interactions, which were not conserved in the AVCP structure. PHCP variants having altered subunit–subunit interactions were more severely destabilized than ones having altered heme‐related interactions. The PHCP structure further revealed a ligand‐binding channel and a penta‐coordinated heme, as observed in the AVCP protein. A spectroscopic study clearly showed that some ligands were bound to the PHCP protein. It is concluded that the dimeric PHCP from the thermophile is effectively stabilized through heme‐related and subunit–subunit interactions with conservation of the ligand‐binding ability.

Thermal stability of cytochrome c' from mesophilic Shewanella amazonensis.

Cytochrome c′ (SACP) from mesophilic Shewanella amazonensis, growing optimally at 37 °C, was thermally more stable than cytochrome c′ (AVCP) from mesophilic Allochromatium vinosum, growing optimally at 25 °C. In contrast, SACP was less stable than cytochrome c′ (PHCP) from thermophilic Hydrogenophilus thermoluteolus, growing optimally at 52 °C. Although only 28% of the SACP amino acid sequence was identical to those of AVCP and PHCP, the latter two being 55% identical, the overall main chain structures of the three cytochromes c′ were similar, and SACP exhibited thermal stability intermediate between those of AVCP and PHCP. For these three proteins, the higher the stability is, the lesser the number of Gly residues in the putative α-helical regions is. Cytochromes c′ including the present three are suitable for examining the protein stabilization mechanisms, because they are structurally similar and available from environments with a wide range of temperatures. Cytochrome c′ from S. amazonensis, growing at 37 °C, exhibited stability intermediate between counterparts from A. vinosum and H. thermoluteolus, growing at 25 and 52 °C, respectively.

High stability of apo-cytochrome c’ from thermophilic Hydrogenophilus thermoluteolus

Apo-cytochomes c without heme are usually unstructured. Here we showed that apo-form of thermophilic Hydrogenophilus thermoluteolus cytochrome c’ (PHCP) was a monomeric protein with high helix content. Apo-PHCP was thermally stable, possibly due to the hydrophobic residues and ion pairs. PHCP is the first example of a structured apo-cytochrome c’, which will expand our view of hemoprotein structure formation.

Pseudomonas aeruginosa cytochrome c551 denaturation by five systematic urea derivatives that differ in the alkyl chain length

Reversible denaturation of Pseudomonas aeruginosa cytochrome c551 (PAc551) could be followed using five systematic urea derivatives that differ in the alkyl chain length, i.e. urea, N-methylurea (MU), N-ethylurea (EU), N-propylurea (PU), and N-butylurea (BU). The BU concentration was the lowest required for the PAc551 denaturation, those of PU, EU, MU, and urea being gradually higher. Furthermore, the accessible surface area difference upon PAc551 denaturation caused by BU was found to be the highest, those by PU, EU, MU, and urea being gradually lower. These findings indicate that urea derivatives with longer alkyl chains are stronger denaturants. In this study, as many as five systematic urea derivatives could be applied for the reversible denaturation of a single protein, PAc551, for the first time, and the effects of the alkyl chain length on protein denaturation were systematically verified by means of thermodynamic parameters. Graphical abstract PAc551 denaturation by five systematic urea derivatives. N-butylurea with the longest alkyl chain is the strongest.

Commonly stabilized cytochromes c from deep-sea Shewanella and Pseudomonas

Abstract Two cytochromes c5 (SBcytc and SVcytc) have been derived from Shewanella living in the deep-sea, which is a high pressure environment, so it could be that these proteins are more stable at high pressure than at atmospheric pressure, 0.1 MPa. This study, however, revealed that SBcytc and SVcytc were more stable at 0.1 MPa than at higher pressure. In addition, at 0.1–150 MPa, the stability of SBcytc and SVcytc was higher than that of homologues from atmospheric-pressure Shewanella, which was due to hydrogen bond formation with the heme in the former two proteins. This study further revealed that cytochrome c551 (PMcytc) of deep-sea Pseudomonas was more stable than a homologue of atmospheric-pressure Pseudomonas aeruginosa, and that specific hydrogen bond formation with the heme also occurred in the former. Although SBcytc and SVcytc, and PMcytc are phylogenetically very distant, these deep-sea cytochromes c are commonly stabilized through hydrogen bond formation. Deep-sea cytochromes c are commonly stabilized through hydrogen bond formation.

Stability and function of a thermophilic cytochrome c

P randomization is the technology that lies behind Isogenica’s ColibraTM offering. It is a defined saturation mutagenesis process that delivers precision control of both identity and relative ratio of amino acids at specified locations within a protein/ antibody library. Thus unwanted amino acids such as cysteine and methionine can be eliminated from libraries because no constraints are imposed by the genetic code. Moreover, the process is non-degenerate, which means that encoding DNA libraries are as small as is physically possible. ProxiMAX relies on a process of saturation cycling comprising ligation, amplification and digestion for each cycle and is the science behind the commercial ColibraTM technology. Currently focused on antibody libraries but with achieved diversities of >99% (6 & 11 saturated codons) and the potential to generate libraries of up to 1014 components, we contest that ProxiMAX randomization is a vital tool in engineering any protein library of the highest quality. This presentation will examine the development of the ProxiMAX process and give examples of libraries created to date.T gondii is a protozoan parasite of medical and veterinary relevance responsible for toxoplasmosis in humans. As there is currently no vaccine available for human, the identification of good target candidates for future drug development is urgently required. A recent proteomic analysis of partially sporulated oocysts of T. gondii showed that oocyctes have a greater capability of de novo amino acid biosynthesis, shedding light on a stage-specific subset of proteins whose functional profile is consistent with the oocyst need to resist various environmental stresses. Among these putative oocyst/sporozoite-specific proteins, three enzymes involved in cysteine metabolism, i.e., cystathionine β-synthase, cystathionine γ-lyase (CGL) and cysteine synthase, were found. However, despite the central metabolic roles of these enzymes, the functionality of none of them has so far been investigated. Herein, CGL from T. gondii (TgCGL) has been cloned, expressed and physiochemically and enzymatically characterized. The purified TgCGL is a functional enzyme which splits L-cystathionine almost exclusively at the CγS bond to yield L-cysteine. This finding likely implies that the reverse transsulfuration pathway is operative in the parasite. The enzyme displays only marginal reactivity toward L-cysteine, which is also a mixed-type inhibitor of TgCGL activity, therefore indicating a tight regulation of cysteine intracellular levels in the parasite. Structure-guided homology modelling revealed two striking amino acid differences between human and parasite CGL active sites (Glu59 and Ser340 in human to Ser77 and Asn360 in toxoplasma). Mutation of these two residues to the corresponding residues in human revealed their importance in modulating both substrate and reaction specificity of the parasitic enzyme. Our findings might have far-reaching implications for the use of TgCGL as anti-toxoplasmosis drug target.

Stabilization of mesophilic Allochromatium vinosum cytochrome c′ through specific mutations modeled by a thermophilic homologue

Abstract AVCP cytochrome c′ from mesophilic Allochromatium vinosum exhibits lower stability than a thermophilic counterpart, Hydrogenophilus thermoluteolus cytochrome c′ (PHCP), in which the six specific amino acid residues that are not conserved in AVCP are responsible for its stability. Here we measured the stability of AVCP variants carrying these specific residues instead of the original AVCP ones. Among the six single AVCP variants, all of which formed a dimeric structure similar to that of the wild-type, three were successfully stabilized compared with the wild-type, while one showed lower stability than the wild-type. In addition, the most stabilized and destabilized AVCP variants could bind CO, similar to the wild-type. These results indicated that mesophilic AVCP could be stabilized through specific three mutations modeled by the thermophilic counterpart, PHCP, without changing the CO binding ability. Mesophilic AVCP could be stabilized through mutations.