Insights into protein adaptation to a saturated salt environment from the crystal structure of a halophilic 2Fe-2S ferredoxin

Frolow, Felix; Harel, Michal; Sussman, Joel L.; Mevarech, Moshe; Shoham, Menachem

doi:10.1038/nsb0596-452

Cited by 215 publications

(214 citation statements)

References 20 publications

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“…A similar structural modification has been reported for the archaeal ferredoxin from Haloarcula marismortui, an extreme halophile [26]. Its N-terminal 30 residues form a loop rich in acidic amino acids and bind a potassium ion in order to adapt to a highly salty environment.…”

Section: Discussionsupporting

confidence: 64%

Zinc and an N‐terminal extra stretch of the ferredoxin from a thermoacidophilic archaeon stabilize the molecule at high temperature

Kojoh

Matsuzawa

Wakagi

1999

European Journal of Biochemistry

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Ferredoxin from the thermoacidophilic archaeon Sulfolobus sp. strain 7 has a 36-residue extra domain at its N-terminus and a 67-residue core domain carrying two iron±sulfur clusters. A zinc ion is held at the interface of the two domains through tetrahedral coordination of three histidine residues (26, 219 and 234) and one aspartic acid residue (276) [Fujii, T., Hata, Y., Oozeki, M., Moriyama, H., Wakagi, T., Tanaka, N. & Oshima, T. (1997) Biochemistry 36, 1505±1513]. To elucidate the roles of the novel zinc ion and the extra N-terminal domain, a series of truncated mutants was constructed: G1, V12, S17, G23, L31 and V38, which lack residues 0, 11, 16, 22, 30 and 37 starting from the N-terminus, respectively. A mutant with two histidine residues each replaced by an alanine residue, H16A/H19A, was also constructed. All the mutant ferredoxins had two iron±sulfur clusters, while zinc was retained only in G1 and V12. The thermal stability of the proteins was investigated by monitoring A 408 ; the melting temperature (T m ) was <109 8C for the natural ferredoxin, <109 8C for G1, 97.6 8C for V12, 89.0 8C for S17, 89.2 8C for G23, 89.3 8C for L31, 82.1 8C for V38, and 89.4 8C for H16A/H19A. K m and V max values of 2-oxoglutarate:ferredoxin oxidoreductase for natural ferredoxin, G1, S17 and L31 were similar, suggesting that electron-accepting activities were not affected by the deletion. The combination of CD and fluorescent spectroscopic analyses with truncated mutant S17 indicated that not only the clusters but also the secondary and tertiary structures were simultaneously degraded at a T m around 89 8C. These results unequivocally demonstrate that the zinc ion and certain parts, but not all, of the extra sequence stretch in the N-terminal domain are responsible not for function but for thermal stabilization of the molecule.Keywords: archaea; ferredoxin; stability; zinc.Ferredoxin is a small protein widely distributed among all living organisms, with one or more redox center(s) composed of acid-labile sulfur and non-heme iron participating in various oxidation±reduction processes in the cell [1,2]. Archaeal ferredoxins are not classified into a simple`archaeal type', but into different categories based on both the clusters and amino-acid sequences; the ferredoxins from thermoacidophilic archaea belong to the 7Fe-bicluster type, those from anaerobic hyperthermophilic archaea to the 4Fe-monocluster type, those from extremely halophilic archaea to the 2Fe-type, and so on [2,3]. It is, however, striking that they play a common role as electron acceptors in the CoA-dependent oxidative decarboxylation of 2-oxoacids, which is seldom found in eubacteria or eukaryotes [4]. In contrast with the widely found 2-oxoacid dehydrogenase multienzyme complex with NAD(P) as an electron acceptor [5], 2-oxoacid:ferredoxin oxidoreductase (OFOR) is a very simple enzyme comprising one to four subunits, with a total molecular mass of around 103±260 kDa [6,7].The primary structure, comprising 103 amino acid residues, of the ferredoxin from the...

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Section: Discussionsupporting

confidence: 64%

Zinc and an N‐terminal extra stretch of the ferredoxin from a thermoacidophilic archaeon stabilize the molecule at high temperature

Kojoh

Matsuzawa

Wakagi

1999

European Journal of Biochemistry

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“…organic solvents) that are used in industrial processes 57 . The main finding of such studies are that the surfaces of proteins from halophiles contain an excess of acidic residues presumably due to their superior water-binding abilities 56 which would help prevent poor protein solubility in such highly dehydrating conditions [58][59][60] . Other studies have also observed a concomitant reduction in lysine content in proteins from halophiles 61; 62 .…”

Section: Proteins From Halophilic Organisms Show An Increased Amount mentioning

confidence: 99%

Amino Acid Contribution to Protein Solubility: Asp, Glu, and Ser Contribute more Favorably than the other Hydrophilic Amino Acids in RNase Sa

Treviño

Scholtz

Pace

2007

Journal of Molecular Biology

215

178

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SUMMARYPoor protein solubility is a common problem in high resolution structural studies, formulation of protein pharmaceuticals, and biochemical characterization of proteins. One popular strategy to improve protein solubility is to use site-directed mutagenesis to make hydrophobic to hydrophilic mutations on the protein surface. However, a systematic investigation of the relative contributions of all twenty amino acids to protein solubility has not been done. Here, twenty variants at the completely solvent-exposed position 76 of Ribonuclease (RNase) Sa are made to compare the contributions of each amino acid. Stability measurements were also made for these variants, which occur at the i+1 position of a type II β-turn. Solubility measurements in ammonium sulfate solutions were made at high positive net charge, low net charge, and high negative net charge. Surprisingly, there was a wide range of contributions to protein solubility even among the hydrophilic amino acids. The results suggest that aspartic acid, glutamic acid, and serine contribute significantly more favorably than the other hydrophilic amino acids especially at high net charge. Therefore, to increase protein solubility, asparagine, glutamine, or threonine should be replaced with aspartic acid, glutamic acid or serine.

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“…Circular permutation of the secondary structure units helped identify previously unrecognized homologs, such as the mercury transporter protein MerP (26). This versatility suggests that a fundamental understanding of the fold's dynamical properties may facilitate the design of proteins with tightly regulated, and unique, functional roles (27). The development of "modular" proteins that can be artificially assembled into large functional complexes is also at the heart of modern synthetic biology (28).…”

mentioning

confidence: 99%

Allostery in the ferredoxin protein motif does not involve a conformational switch

Nechushtai

Lammert²,

Michaeli³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Regulation of protein function via cracking, or local unfolding and refolding of substructures, is becoming a widely recognized mechanism of functional control. Oftentimes, cracking events are localized to secondary and tertiary structure interactions between domains that control the optimal position for catalysis and/or the formation of protein complexes. Small changes in free energy associated with ligand binding, phosphorylation, etc., can tip the balance and provide a regulatory functional switch. However, understanding the factors controlling function in single-domain proteins is still a significant challenge to structural biologists. We investigated the functional landscape of a single-domain planttype ferredoxin protein and the effect of a distal loop on the electron-transfer center. We find the global stability and structure are minimally perturbed with mutation, whereas the functional properties are altered. Specifically, truncating the L1,2 loop does not lead to large-scale changes in the structure, determined via X-ray crystallography. Further, the overall thermal stability of the protein is only marginally perturbed by the mutation. However, even though the mutation is distal to the iron-sulfur cluster (∼20 Å), it leads to a significant change in the redox potential of the ironsulfur cluster (57 mV). Structure-based all-atom simulations indicate correlated dynamical changes between the surface-exposed loop and the iron-sulfur cluster-binding region. Our results suggest intrinsic communication channels within the ferredoxin fold, composed of many short-range interactions, lead to the propagation of long-range signals. Accordingly, protein interface interactions that involve L1,2 could potentially signal functional changes in distal regions, similar to what is observed in other allosteric systems.electron transfer | functional energy landscape | iron-sulfur proteins | protein folding O ver the last several decades, our understanding of protein function has evolved from a rather static perspective, where signaling and function have been understood through surface complementarity arguments, such as the "lock and key" paradigm (1, 2), to a more dynamic view where protein conformational fluctuations are inextricably linked to function (3). As our understanding of protein dynamics expands, we are revealing many mechanisms by which proteins exploit conformational fluctuations to perform cellular function. In multidomain proteins, relative repositioning of domains is often linked to their levels of activity. For example, large-scale domain rearrangements in the four-domain Src kinase (4) and C-terminal Src kinase (5, 6) lead to these proteins being in so-called "on" or "off" states, and functional regulation may be obtained by adjusting the balance between these conformations (7). In proteins such as adenylate kinase, domain rearrangements can be rate limiting during each round of catalysis (8), where the enzyme cycles between ligandcompetent and ligand-release conformations. Because the kinetics of these tra...

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Insights into protein adaptation to a saturated salt environment from the crystal structure of a halophilic 2Fe-2S ferredoxin

Cited by 215 publications

References 20 publications

Zinc and an N‐terminal extra stretch of the ferredoxin from a thermoacidophilic archaeon stabilize the molecule at high temperature

Zinc and an N‐terminal extra stretch of the ferredoxin from a thermoacidophilic archaeon stabilize the molecule at high temperature

Amino Acid Contribution to Protein Solubility: Asp, Glu, and Ser Contribute more Favorably than the other Hydrophilic Amino Acids in RNase Sa

Allostery in the ferredoxin protein motif does not involve a conformational switch

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