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Constantinos E Vorgias - One of the best experts on this subject based on the ideXlab platform.
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high resolution x ray structure of the dna binding Protein hu from the hyper Thermophilic thermotoga maritima and the determinants of its thermostability
Extremophiles, 2003Co-Authors: Evangelos Christodoulou, Wojciech Rypniewski, Constantinos E VorgiasAbstract:The histone-like DNA-binding Proteins (HU) are a convenient model for studying factors affecting thermostability because of their relatively simple, easily comparable structures, their common function, and their presence in organisms of widely differing thermostability. We report the determination of the high-resolution structure (1.53 A) at 273 K and 100 K of the HU Protein from the hyper-Thermophilic eubacterium Thermotoga maritima(HU Tmar, T(m)=80.5 degrees C). The structural data presented clearly show that the HU Tmar has a fold similar to its Thermophilic homologue HU from Bacillus stearothermophilus (HU Bst). Based on primary structure analysis, as well as on the results of mutational analysis of HU Bst ( T(m)=61.6 degrees C) and Bacillus subtilis (HU Bsu, T(m)=39.7 degrees C), we have designed and produced several single and combined mutations to study their effect on the thermostability of the recombinant HU Tmar. Among others, the triplet mutant HU Tmar-G15E/E34D/V42I ( T(m)=35.9 degrees C) has converted the extreme Thermophilic Protein HU Tmar to mesophilic, like HU Bsu. In an attempt to analyze the various mutants of HU Tmar, we crystallized the point mutation HU Tmar-E34D, in which Glu34 was replaced by Asp, similar to the mesophilic HU Bsu. The mutant has T(m)=72.9 degrees C, as measured by circular dichroism, 7.6 degrees C lower than the wild type. The crystal structure of HU Tmar-E34D was determined at 100 K and refined at 1.72 A resolution. A comparison with the wild-type structures clearly shows that two hydrogen bonds have been disrupted between Glu34 from one subunit and Thr13 from the other subunit, and vice versa. Our analysis points to this as the prime cause of the destabilization compared to the wild type. The three new structures were compared, together with the X-ray structure of a similar Protein, HU Bst, with the aim of relating their structural properties and different thermal stability. The presented results show that the HU Tmar Protein achieves its stability by employing a dual strategy. On the one hand, we observe local hydrophobic interactions, which stabilize the secondary structure elements, and on the other hand, electrostatic interactions between side chains.
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the thermostability of dna binding Protein hu from mesophilic Thermophilic and extreme Thermophilic bacteria
Extremophiles, 2002Co-Authors: Evangelos Christodoulou, Constantinos E VorgiasAbstract:Based on primary structure comparison between four highly homologous DNA-binding Proteins (HUs) displaying differential thermostability, we have employed in vitro site-directed mutagenesis to decipher their thermostability mechanism at the molecular level. The contribution of the 11 amino acids that differ between the Thermophilic HUBst from Bacillus stearothermophilus (Tm=61.6°C) and the mesophilic HUBsu from Bacillus subtilis (Tm=39.7°C) was evaluated by replacing these amino acids in HUBst with their mesophilic counterparts. Among 11 amino acids, three residues, Gly-15, Glu-34, and Val-42, which are highly conserved in the Thermophilic HUs, have been found to be responsible for the thermostability of HUBst. These amino acids in combination (HUBst-G15E/E34D/V42I) reduce the thermostability of the Protein (Tm=45.1°C) at the level of its mesophilic homologue HUBsu. By replacing these amino acids in HUBsu with their Thermophilic counterparts, the HUBsu-E15G/D34E/I42V mutant was generated with thermostability (Tm=57.8°C) at the level of Thermophilic HUBst. Employing the same strategy, we generated several mutants in the extremely Thermophilic HUTmar from Thermotoga maritima (Tm=80.5°C), and obtained data consistent with the previous results. The triplet mutant HUTmar-G15E/E34D/V42I (Tm=35.9°C) converted the extremely Thermophilic Protein HUTmar to mesophilic. The various forms of HU Proteins were overproduced in Escherichia coli, highly purified, and the thermostability of the mutants confirmed by circular dichroism spectroscopy. The results presented here were elucidated on the basis of the X-ray structure of HUBst and HUTmar (our unpublished results), and their mechanism was proposed at the molecular level. The results clearly show that three individual local interactions located at the helix-turn-helix part of the Protein are responsible for the stability of HU Proteins by acting cooperatively in a common mechanism for thermostability.
Susan Marqusee - One of the best experts on this subject based on the ideXlab platform.
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structure stability and folding of ribonuclease h1 from the moderately Thermophilic chlorobium tepidum comparison with Thermophilic and mesophilic homologues
Biochemistry, 2009Co-Authors: Kathleen Ratcliff, Jacob E Corn, Susan MarquseeAbstract:Proteins from Thermophilic organisms are able to function under conditions that render a typical mesophilic Protein inactive. Pairwise comparisons of homologous mesophilic and Thermophilic Proteins can help to identify the energetic features of a Protein’s energy landscape that lead to such thermostability. Previous studies of bacterial ribonucleases H (RNases H) from the thermophile Thermus thermophilus and the mesophile Escherichia coli revealed that the thermostability arises in part from an unusually low change in heat capacity upon unfolding (ΔCp) for the Thermophilic Protein [Hollien, J., and Marqusee, S. (1999) Biochemistry 38, 3831−3836]. Here, we have further examined how nearly identical Proteins can adapt to different thermal constraints by adding a moderately Thermophilic homologue to the previously characterized mesophilic and Thermophilic pair. We identified a putative RNase H from Chlorobium. tepidum and demonstrated that it is an active RNase H and adopts the RNase H fold. The moderately t...
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contributions of folding cores to the thermostabilities of two ribonucleases h
Protein Science, 2009Co-Authors: Srebrenka Robic, James M Berger, Susan MarquseeAbstract:To investigate the contribution of the folding cores to the thermodynamic stability of RNases H, we used rational design to create two chimeras composed of parts of a Thermophilic and a mesophilic RNase H. Each chimera combines the folding core from one parent Protein and the remaining parts of the other. Both chimeras form active, well-folded RNases H. Stability curves, based on CD-monitored chemical denaturations, show that the chimera with the Thermophilic core is more stable, has a higher midpoint of thermal denaturation, and a lower change in heat capacity (ΔCp) upon unfolding than the chimera with the mesophilic core. A possible explanation for the low ΔCp of both the parent Thermophilic RNase H and the chimera with the Thermophilic core is the residual structure in the denatured state. On the basis of the studied parameters, the chimera with the Thermophilic core resembles a true Thermophilic Protein. Our results suggest that the folding core plays an essential role in conferring thermodynamic parameters to RNases H.
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role of residual structure in the unfolded state of a Thermophilic Protein
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Srebrenka Robic, Mercedes Guzmancasado, Jose M Sanchezruiz, Susan MarquseeAbstract:Ribonucleases H from the Thermophilic bacterium Thermus thermophilus and the mesophile Escherichia coli demonstrate a dramatic and surprising difference in their change in heat capacity upon unfolding (ΔCp°). The lower ΔCp° of the Thermophilic Protein directly contributes to its higher thermal denaturation temperature ( T m ). We propose that this ΔCp° difference originates from residual structure in the unfolded state of the Thermophilic Protein; we verify this hypothesis by using a mutagenic approach. Residual structure in the unfolded state may provide a mechanism for balancing a high T m with the optimal thermodynamic stability for a Protein9s function. Structure in the unfolded state is shown to differentially affect the thermodynamic profiles of Thermophilic and mesophilic Proteins.
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comparison of the folding processes of t thermophilus and e coli ribonucleases h
Journal of Molecular Biology, 2002Co-Authors: Julie Hollien, Susan MarquseeAbstract:Abstract In order to examine how the stabilization of Thermophilic Proteins affects their folding, we have characterized the folding process of Thermus thermophilus ribonuclease H using circular dichroism, fluorescence, and pulse-labeling hydrogen exchange. Like its homolog from Escherichia coli , this Thermophilic Protein populates a partially folded kinetic intermediate within the first few milliseconds of folding. The structure of this intermediate is similar to that of E. coli RNase H and corresponds remarkably well to a partially folded form that is populated at low levels in the native state of the Protein. Proline isomerization appears to partly limit the folding of the Thermophilic but not the mesophilic Protein. Lastly, unlike other Thermophilic Proteins, which unfold much more slowly than their mesophilic counterparts, T. thermophilus RNase H folds and unfolds with overall rates similar to those of E. coli RNase H.
Evangelos Christodoulou - One of the best experts on this subject based on the ideXlab platform.
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high resolution x ray structure of the dna binding Protein hu from the hyper Thermophilic thermotoga maritima and the determinants of its thermostability
Extremophiles, 2003Co-Authors: Evangelos Christodoulou, Wojciech Rypniewski, Constantinos E VorgiasAbstract:The histone-like DNA-binding Proteins (HU) are a convenient model for studying factors affecting thermostability because of their relatively simple, easily comparable structures, their common function, and their presence in organisms of widely differing thermostability. We report the determination of the high-resolution structure (1.53 A) at 273 K and 100 K of the HU Protein from the hyper-Thermophilic eubacterium Thermotoga maritima(HU Tmar, T(m)=80.5 degrees C). The structural data presented clearly show that the HU Tmar has a fold similar to its Thermophilic homologue HU from Bacillus stearothermophilus (HU Bst). Based on primary structure analysis, as well as on the results of mutational analysis of HU Bst ( T(m)=61.6 degrees C) and Bacillus subtilis (HU Bsu, T(m)=39.7 degrees C), we have designed and produced several single and combined mutations to study their effect on the thermostability of the recombinant HU Tmar. Among others, the triplet mutant HU Tmar-G15E/E34D/V42I ( T(m)=35.9 degrees C) has converted the extreme Thermophilic Protein HU Tmar to mesophilic, like HU Bsu. In an attempt to analyze the various mutants of HU Tmar, we crystallized the point mutation HU Tmar-E34D, in which Glu34 was replaced by Asp, similar to the mesophilic HU Bsu. The mutant has T(m)=72.9 degrees C, as measured by circular dichroism, 7.6 degrees C lower than the wild type. The crystal structure of HU Tmar-E34D was determined at 100 K and refined at 1.72 A resolution. A comparison with the wild-type structures clearly shows that two hydrogen bonds have been disrupted between Glu34 from one subunit and Thr13 from the other subunit, and vice versa. Our analysis points to this as the prime cause of the destabilization compared to the wild type. The three new structures were compared, together with the X-ray structure of a similar Protein, HU Bst, with the aim of relating their structural properties and different thermal stability. The presented results show that the HU Tmar Protein achieves its stability by employing a dual strategy. On the one hand, we observe local hydrophobic interactions, which stabilize the secondary structure elements, and on the other hand, electrostatic interactions between side chains.
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the thermostability of dna binding Protein hu from mesophilic Thermophilic and extreme Thermophilic bacteria
Extremophiles, 2002Co-Authors: Evangelos Christodoulou, Constantinos E VorgiasAbstract:Based on primary structure comparison between four highly homologous DNA-binding Proteins (HUs) displaying differential thermostability, we have employed in vitro site-directed mutagenesis to decipher their thermostability mechanism at the molecular level. The contribution of the 11 amino acids that differ between the Thermophilic HUBst from Bacillus stearothermophilus (Tm=61.6°C) and the mesophilic HUBsu from Bacillus subtilis (Tm=39.7°C) was evaluated by replacing these amino acids in HUBst with their mesophilic counterparts. Among 11 amino acids, three residues, Gly-15, Glu-34, and Val-42, which are highly conserved in the Thermophilic HUs, have been found to be responsible for the thermostability of HUBst. These amino acids in combination (HUBst-G15E/E34D/V42I) reduce the thermostability of the Protein (Tm=45.1°C) at the level of its mesophilic homologue HUBsu. By replacing these amino acids in HUBsu with their Thermophilic counterparts, the HUBsu-E15G/D34E/I42V mutant was generated with thermostability (Tm=57.8°C) at the level of Thermophilic HUBst. Employing the same strategy, we generated several mutants in the extremely Thermophilic HUTmar from Thermotoga maritima (Tm=80.5°C), and obtained data consistent with the previous results. The triplet mutant HUTmar-G15E/E34D/V42I (Tm=35.9°C) converted the extremely Thermophilic Protein HUTmar to mesophilic. The various forms of HU Proteins were overproduced in Escherichia coli, highly purified, and the thermostability of the mutants confirmed by circular dichroism spectroscopy. The results presented here were elucidated on the basis of the X-ray structure of HUBst and HUTmar (our unpublished results), and their mechanism was proposed at the molecular level. The results clearly show that three individual local interactions located at the helix-turn-helix part of the Protein are responsible for the stability of HU Proteins by acting cooperatively in a common mechanism for thermostability.
Peter Zavodszky - One of the best experts on this subject based on the ideXlab platform.
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structural differences between mesophilic moderately Thermophilic and extremely Thermophilic Protein subunits results of a comprehensive survey
Structure, 2000Co-Authors: Andras Szilagyi, Peter ZavodszkyAbstract:Results: In order to reveal the general evolutionary strategy for changing the heat stability of Proteins, a non-redundant data set was compiled comprising all high-quality structures of Thermophilic Proteins and their mesophilic homologues from the Protein Data Bank. The selection (quality) criteria were met by 64 mesophilic and 29 Thermophilic Protein subunits, representing 25 Protein families. From the atomic coordinates, 13 structural parameters were calculated, compared and evaluated using statistical methods. This study is distinguished from earlier ones by the strict quality control of the structures used and the size of the data set. Conclusions: Different Protein families adapt to higher temperatures by different sets of structural devices. Regarding the structural parameters, the only generally observed rule is an increase in the number of ion pairs with increasing growth temperature. Other parameters show just a trend, whereas the number of hydrogen bonds and the polarity of buried surfaces exhibit no clear-cut tendency to change with growth temperature. Proteins from extreme thermophiles are stabilized in different ways to moderately Thermophilic ones. The preferences of these two groups are different with regards to the number of ion pairs, the number of cavities, the polarity of exposed surface and the secondary structural composition.
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relationship between thermal stability and 3 d structure in a homology model of 3 isopropylmalate dehydrogenase from escherichia coli
Protein Engineering, 1996Co-Authors: Csaba Magyar, Andras Szilagyi, Peter ZavodszkyAbstract:: To reveal the structural basis of the increased thermal stability of 3-isopropylmalate dehydrogenase (IPMDH) from Thermus thermophilus, an extreme thermophile, the homology-based structural model of one mesophilic (Escherichia coli) counterpart, was constructed. Both IPMDHs are homodimeric Proteins. We built a model of one subunit using the 3-D structures of the Th. thermophilus IPMDH and the homologous E.coli isocitrate dehydrogenase. Energy minimization and molecular dynamics simulated annealing were performed on the dimer, including a surrounding solvation shell. No serious errors were detected in the refined model using the 3-D profile method. The resulting structure was scrutinized and compared with the structure of the Th.thermophilus IPMDH. Significant differences were found in the non-specific interactions including the hydrophobic effect. The model predicts a higher number of ion pairs in the Th.thermophilus than in the E.coli enzyme. An increase was observed in the stabilities of alpha-helical regions in the Thermophilic Protein. The preliminary X-ray coordinates of the E.coli IPMDH were received after the completion of this work, allowing an assessment of the model in terms of the X-ray structure. The comparison proved that most of the structural features underlying the stability differences between the two enzymes were predicted correctly.
Fabio Sterpone - One of the best experts on this subject based on the ideXlab platform.
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Interface matters: the stiffness route to stability of a Thermophilic tetrameric malate dehydrogenase.
PLoS ONE, 2014Co-Authors: Maria Kalimeri, Eric Girard, Dominique Madern, Fabio SterponeAbstract: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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how conformational flexibility stabilizes the hyperThermophilic elongation factor g domain
Journal of Physical Chemistry B, 2013Co-Authors: Maria Kalimeri, Obaidur Rahaman, Simone Melchionna, Fabio SterponeAbstract:Proteins from Thermophilic organisms are stable and functional well above ambient temperature. Understanding the molecular mechanism underlying such a resistance is of crucial interest for many technological applications. For some time, thermal stability has been assumed to correlate with high mechanical rigidity of the Protein matrix. In this work we address this common belief by carefully studying a pair of homologous G-domain Proteins, with their melting temperatures differing by 40 K. To probe the thermal-stability content of the two Proteins we use extensive simulations covering the microsecond time range and employ several different indicators to assess the salient features of the conformational landscape and the role of internal fluctuations at ambient condition. At the atomistic level, while the magnitude of fluctuations is comparable, the distribution of flexible and rigid stretches of amino-acids is more regular in the Thermophilic Protein causing a cage-like correlation of amplitudes along the ...