Thermostability of multidomain proteins: Elongation factors EF‐Tu from <i>Escherichia coli</i> and <i>Bacillus stearothermophilus</i> and their chimeric forms

Šanderová, Hana

doi:10.1110/ps.03272504

Cited by 23 publications

(16 citation statements)

References 42 publications

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“…The eEF1A⅐GTP complex was more stable than the eEF1A⅐GDP complex. This finding is consistent with some G-proteins being more thermostable in the GTP-bound conformation than in the GDP-bound conformation, which may be explained by the extra interaction with the ␥-phosphate residue of GTP (36,37). The structures of GTP-and GDP-bound ET-Tu show that a large rearrangement occurs upon GTP hydrolysis (36,38).…”

Section: Discussionsupporting

confidence: 84%

Guanine Nucleotide Exchange Factor Independence of the G-protein eEF1A through Novel Mutant Forms and Biochemical Properties

Ozturk¹,

Kinzy²

2008

Journal of Biological Chemistry

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Most G-proteins require a guanine nucleotide exchange factor (GEF) to regulate a variety of critical cellular processes. Interestingly, a small number of G-proteins switch between the active and inactive forms without a GEF. Translation elongation factor 1A (eEF1A) normally requires the GEF eEF1B␣ to accelerate nucleotide dissociation. However, several mutant forms of eEF1A are functional independent of this essential regulator in vivo. GEF-independent eEF1A mutations localize close to the G-protein motifs that are crucial for nucleotide binding. Kinetic analysis demonstrated that reduced GDP affinity correlates with wild type growth and high translation activities of GEFindependent mutants. Furthermore, the mutant forms show an 11-22-fold increase in rates of GDP dissociation from eEF1A compared with the wild type protein. All mutant forms have dramatically enhanced stability at elevated temperatures. This, coupled with data demonstrating that eEF1A is also more stable in the presence of nucleotides, suggests that both the GEF and nucleotide have stabilizing effects on eEF1A. The biochemical properties of these eEF1A mutants provide insight into the mechanism behind GEF-independent G-protein function.GTPases regulate a variety of cellular functions with a conserved mechanism of nucleotide binding and hydrolysis. Signal transduction, control of cell cycle and differentiation during cell division, protein biosynthesis, vesicular trafficking, and translocation of membrane proteins are key cellular processes where GTPases play critical roles. Based on their functional roles, domain structures, or sizes, the superfamily of GTPases can be divided into many families, including small G proteins (Ras GTPase superfamily), heterotrimeric G proteins, and the translation factor family (1). All GTPases share a G-domain with conserved sequence elements, such as switch I and II, the P-loop (phosphate binding loop), and the NKXD element (2). Guanine nucleotides form specific interactions with these sites, which are modulated by G-protein accessory factors to create the switch mechanism between the active and inactive forms (1, 3).GTPase activation factors stimulate GTP hydrolysis, resulting in the inactive GDP-bound form. Guanine nucleotide exchange factors (GEFs) 2 catalyze GDP release by reducing the nucleotide affinity (1). This allows G-proteins to rebind GTP due to a higher cellular concentration of GTP and thus switch to their active form. The GEFs interact with the switch I and II regions while inserting residues close to or into the P-loop and Mg 2ϩ binding site. The insertion of the GEF residues perturbs the interaction surface in the phosphate binding region, resulting in the release of phosphate groups, which in turn causes dissociation of the nucleotide. In contrast to the mechanism of exchange and the G-proteins themselves, GEFs show little to no conservation in sequence or structure (3).The crystal structures of the majority of G-proteins solved in the presence of nucleotides show that the ␤-phosphate of the nucle...

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Section: Discussionsupporting

confidence: 84%

Guanine Nucleotide Exchange Factor Independence of the G-protein eEF1A through Novel Mutant Forms and Biochemical Properties

Ozturk¹,

Kinzy²

2008

Journal of Biological Chemistry

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“…Moreover, the region where this instability is localized is the curved helix ( α 1G- α 2G), an important portion of which is part of the catalytic pocket. This finding indicates that the isolated domains set possibly a baseline in the tetramers' thermal resistance but extra stability is gained by domain-domain interactions [51] .…”

Section: Discussionmentioning

confidence: 94%

Interface Matters: The Stiffness Route to Stability of a Thermophilic Tetrameric Malate Dehydrogenase

2014

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In this work we investigate by computational means the behavior of two orthologous bacterial proteins, a mesophilic and a thermophilic tetrameric malate dehydrogenase (MalDH), at different temperatures. Namely, we quantify how protein mechanical rigidity at different length- and time-scales correlates to protein thermophilicity as commonly believed. In particular by using a clustering analysis strategy to explore the conformational space of the folded proteins, we show that at ambient conditions and at the molecular length-scale the thermophilic variant is indeed more rigid that the mesophilic one. This rigidification is the result of more efficient inter-domain interactions, the strength of which is further quantified via ad hoc free energy calculations. When considered isolated, the thermophilic domain is indeed more flexible than the respective mesophilic one. Upon oligomerization, the induced stiffening of the thermophilic protein propagates from the interface to the active site where the loop, controlling the access to the catalytic pocket, anchors down via an extended network of ion-pairs. On the contrary in the mesophilic tetramer the loop is highly mobile. Simulations at high temperature, could not re-activate the mobility of the loop in the thermophile. This finding opens questions on the similarities of the binding processes for these two homologues at their optimal working temperature and suggests for the thermophilic variant a possible cooperative role of cofactor/substrate.

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“…EF‐Tu is an abundant, highly conserved cellular guanosine triphosphate (GTP)‐protein, occupying a key position in translation and plays an important role in protein biosynthesis (Sanderova et al . ). The down‐regulation of EF‐Tu in our study suggested that protein biosynthesis was inhibited in N‐deplete P. donghaiense cells.…”

Section: Discussionmentioning

confidence: 97%

Proteomic analysis provides new insights into the adaptive response of a dinoflagellate Prorocentrum donghaiense to changing ambient nitrogen

Zhang

et al. 2015

Plant Cell & Environment

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Nitrogen (N) is the major nutrient limiting phytoplankton growth and productivity over large ocean areas. Dinoflagellates are important primary producers and major causative agents of harmful algal blooms in the ocean. However, very little is known about their adaptive response to changing ambient N. Here, we compared the protein profiles of a marine dinoflagellate Prorocentrum donghaiense grown in inorganic N-replete, N-deplete and N-resupplied conditions using 2-D fluorescence differential gel electrophoresis. The results showed that cell density, chlorophyll a and particulate organic N contents presented low levels in N-deplete cells, while particulate organic carbon content and glutamine synthetase (GS) activity maintained high levels. Comparison of the protein profiles of N-replete, N-deplete and N-resupplied cells indicated that proteins involved in photosynthesis, carbon fixation, protein and lipid synthesis were down-regulated, while proteins participating in N reallocation and transport activity were up-regulated in N-deplete cells. High expressions of GS and 60 kDa chaperonin as well as high GS activity in N-deplete cells indicated their central role in N stress adaptation. Overall, in contrast with other photosynthetic eukaryotic algae, P. donghaiense possessed a specific ability to regulate intracellular carbon and N metabolism in response to extreme ambient N deficiency.

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Thermostability of multidomain proteins: Elongation factors EF‐Tu from Escherichia coli and Bacillus stearothermophilus and their chimeric forms

Cited by 23 publications

References 42 publications

Guanine Nucleotide Exchange Factor Independence of the G-protein eEF1A through Novel Mutant Forms and Biochemical Properties

Guanine Nucleotide Exchange Factor Independence of the G-protein eEF1A through Novel Mutant Forms and Biochemical Properties

Interface Matters: The Stiffness Route to Stability of a Thermophilic Tetrameric Malate Dehydrogenase

Proteomic analysis provides new insights into the adaptive response of a dinoflagellate Prorocentrum donghaiense to changing ambient nitrogen

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