Model‐free analysis of a thermophilic Fe7S8 protein compared with a mesophilic Fe4S4 protein

Bertini, Ivano; Luchinat, Claudio; Niikura, Yohei; Presenti, Chiara

doi:10.1002/1097-0134(20001001)41:1<75::aid-prot100>3.3.co;2-u

Cited by 3 publications

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“…In the present study, we have also analyzed the backbone dynamics of both the A. acidocaldarius thioredoxin and its K18G/R82E mutant in a wide temperature range from 25 °C up to 65 °C, so including the growth temperature of the bacterium, which is between 55 and 65 °C (). Even if many studies have been published that report protein backbone dynamics studies as function of temperature ( − ), a minor part of them were focused on the mobility of thermostable proteins analyzed in a range comprising the temperature range of growth of their sources.…”

Section: Discussionmentioning

confidence: 99%

Solution Structure and Backbone Dynamics of the K18G/R82E Alicyclobacillus acidocaldarius Thioredoxin Mutant: A Molecular Analysis of Its Reduced Thermal Stability^,

et al. 2004

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No general strategy for thermostability has been yet established, because the extra stability of thermophiles appears to be the sum of different cumulative stabilizing interactions. In addition, the increase of conformational rigidity observed in many thermophilic proteins, which in some cases disappears when mesophilic and thermophilic proteins are compared at their respective physiological temperatures, suggests that evolutionary adaptation tends to maintain corresponding states with respect to conformational flexibility. In this study, we accomplished a structural analysis of the K18G/R82E Alicyclobacillus acidocaldarius thioredoxin (BacTrx) mutant, which has reduced heat resistance with respect to the thermostable wild-type. Furthermore, we have also achieved a detailed study, carried out at 25, 45, and 65 degrees C, of the backbone dynamics of both the BacTrx and its K18G/R82E mutant. Our findings clearly indicate that the insertion of the two mutations causes a loss of energetically favorable long-range interactions and renders the secondary structure elements of the double mutants more similar to those of the mesophilic Escherichia coli thioredoxin. Moreover, protein dynamics analysis shows that at room temperature the BacTrx, as well as the double mutant, are globally as rigid as the mesophilic thioredoxins; differently, at 65 degrees C, which is in the optimal growth temperature range of A. acidocaldarius, the wild-type retains its rigidity while the double mutant is characterized by a large increase of the amplitude of the internal motions. Finally, our research interestingly shows that fast motions on the pico- to nanosecond time scale are not detrimental to protein stability and provide an entropic stabilization of the native state. This study further confirms that protein thermostability is reached through diverse stabilizing interactions, which have the key role to maintain the structural folding stable and functional at the working temperature.

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Section: Discussionmentioning

confidence: 99%

Solution Structure and Backbone Dynamics of the K18G/R82E Alicyclobacillus acidocaldarius Thioredoxin Mutant: A Molecular Analysis of Its Reduced Thermal Stability^,

et al. 2004

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“…Various experimental methods have been applied to explore protein dynamics adaptation to high temperature. Hydrogen/deuterium (H/D) exchange [22], fluorescence quenching [23], high‐resolution NMR [24] and neutron scattering [25] are probably the major ones. The flexibility of protein molecules is reflected in conformational fluctuations.…”

Section: Experimental Methods Used To Study the Role Of Dynamics In Tmentioning

confidence: 99%

Adaptation to high temperatures through macromolecular dynamics by neutron scattering

Tehei

Zaccaı̈

2007

The FEBS Journal

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Work on the relationship between hyperthermophile protein dynamics, stability and activity is reviewed. Neutron spectroscopy has been applied to measure and compare the macromolecular dynamics of various hyperthermophilic and mesophilic proteins, under different conditions. First, molecular dynamics have been analyzed for the hyperthermophile malate dehydrogenase from Methanococcus jannaschii and a mesophilic homologue, the lactate dehydrogenase from Oryctolagus cunniculus (rabbit) muscle. The neutron scattering approach has provided independent measurements of the global flexibility and structural resilience of each protein, and it has been demonstrated that macromolecular dynamics represents one of the molecular mechanisms of thermoadaptation. The resilience was found to be higher for the hyperthermophilic protein, thus ensuring similar flexibilities in both enzymes at their optimal activity temperature. Second, the neutron method has been developed to quantify the average macromolecular flexibility and resilience within the natural crowded environment of the cell, and mean macromolecular motions have been measured in vivo in psychrophile, mesophile, thermophile and hyperthermophile bacteria. The macromolecular resilience in bacteria was found to increase with adaptation to high temperatures, whereas flexibility was maintained within narrow limits, independent of physiological temperature for all cells in their active state. Third, macromolecular motions have been measured in free and immobilized dihydrofolate reductase from Escherichia coli. The immobilized mesophilic enzyme has increased stability and decreased activity, so that its properties are changed to resemble those of a thermophilic enzyme. Quasi‐elastic neutron scattering measurements have also been performed to probe the protein motions. Compared to the free enzyme, the average height of the activation free energy barrier to local motions was found to be increased by 0.54 kcal·mol−1 in the immobilized dihydrofolate reductase, a value that is of the same order as expected from the theoretical rate equation.

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“…We discuss below the results obtained for the axially symmetric case only. By analyzing the τ m values of a group of proteins (6,64,(69)(70)(71)(72), determined experimentally by NMR, versus their molecular volumes (at normalized temperatures), we found that the present τ m values are higher than expected. Since the protein tends to aggregate, care is taken to avoid this possibility.…”

Section: A Comment On the Possible Effects Of Unpaired Electronsmentioning

confidence: 65%

“…Measurement of 15 N relaxation rates has become an important means of studying the backbone dynamics of proteins in solution ( − ). They can be conveniently analyzed on the millisecond to microsecond and picosecond to nanosecond time scales ( − ).…”

mentioning

confidence: 99%

¹⁵N Backbone Dynamics of Ferricytochrome b₅₆₂: Comparison with the Reduced Protein and the R98C Variant

et al. 2001

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The backbone dynamics of ferricytochrome b(562), a four-helix bundle protein from Escherichia coli, have been studied by NMR spectroscopy. The consequences of the introduction of a c-type thioether linkage between the heme and protein and the reduction to the ferrous cytochrome have also been analyzed. (15)N relaxation rates R(1) and R(2) and (1)H-(15)N NOEs were measured at proton Larmor frequencies of 500 and 600 MHz for the oxidized and reduced protein as well as for the oxidized R98C variant. In the latter protein, an "artificial" thioether covalent bond has been introduced between the heme group and the protein frame [Arnesano, F., Banci, L., Bertini, I., Ciofi-Baffoni, S., de Lumley Woodyear, T., Johnson, C. M., and Barker, P. D. (2000) Biochemistry 39, 1499-1514]. The (15)N relaxation data were analyzed with the ModelFree protocol, and the mobility parameters on the picosecond to nanosecond time scale were compared for the three species. The three forms are rather rigid as a whole, with average generalized order parameters values of 0.87 +/- 0.08 (oxidized cytochrome b(562)), 0.84 +/- 0.07 (reduced cytochrome b(562)), and 0.85 +/- 0.07 (oxidized R98C cytochrome b(562)), indicating similar mobility for each system. Lower order parameters (S(2)) are found for residues belonging to loops 1 and 2. Higher mobility, as indicated by lower order parameters, is found for heme binding helices alpha 1 and alpha 4 in the R98C variant with respect to the wild-type protein. The analysis requires a relatively long rotational correlation time (tau(m) = 9.6 ns) whose value is accounted for on the basis of the anisotropy of the molecular shape and the high phosphate concentration needed to ensure the occurrence of monomer species. A parallel study of motions in the millisecond to microsecond time scale has also been performed on oxidized wild-type and R98C cytochrome b(562). In a CPMG experiment, decay rates were analyzed in the presence of spin-echo pulse trains of variable spacing. The dynamic behavior on this time scale is similar to that observed on the sub-nanosecond time scale, showing an increased mobility in the residues connected to the heme ligands in the R98C variant. It appears that the increased protein stability of the variant, established previously, is not correlated with an increase in rigidity.

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Model‐free analysis of a thermophilic Fe7S8 protein compared with a mesophilic Fe4S4 protein

Cited by 3 publications

References 0 publications

Solution Structure and Backbone Dynamics of the K18G/R82E Alicyclobacillus acidocaldarius Thioredoxin Mutant: A Molecular Analysis of Its Reduced Thermal Stability^,

Solution Structure and Backbone Dynamics of the K18G/R82E Alicyclobacillus acidocaldarius Thioredoxin Mutant: A Molecular Analysis of Its Reduced Thermal Stability^,

Adaptation to high temperatures through macromolecular dynamics by neutron scattering

¹⁵N Backbone Dynamics of Ferricytochrome b₅₆₂: Comparison with the Reduced Protein and the R98C Variant

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Model‐free analysis of a thermophilic Fe7S8 protein compared with a mesophilic Fe4S4 protein

Cited by 3 publications

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Solution Structure and Backbone Dynamics of the K18G/R82E Alicyclobacillus acidocaldarius Thioredoxin Mutant: A Molecular Analysis of Its Reduced Thermal Stability,

Solution Structure and Backbone Dynamics of the K18G/R82E Alicyclobacillus acidocaldarius Thioredoxin Mutant: A Molecular Analysis of Its Reduced Thermal Stability,

Adaptation to high temperatures through macromolecular dynamics by neutron scattering

15N Backbone Dynamics of Ferricytochrome b562: Comparison with the Reduced Protein and the R98C Variant

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Solution Structure and Backbone Dynamics of the K18G/R82E Alicyclobacillus acidocaldarius Thioredoxin Mutant: A Molecular Analysis of Its Reduced Thermal Stability^,

Solution Structure and Backbone Dynamics of the K18G/R82E Alicyclobacillus acidocaldarius Thioredoxin Mutant: A Molecular Analysis of Its Reduced Thermal Stability^,

¹⁵N Backbone Dynamics of Ferricytochrome b₅₆₂: Comparison with the Reduced Protein and the R98C Variant