PubMed Health⌕ Search
Home/Archive/PubMed

PubMed · 16106445

Temperature-dependent structural and functional features of a hyperthermostable enzyme using elastic neutron scattering.

Sotirios Koutsopoulos·John van der Oost·Willem Norde
View original source ↗Export metadata ↓

Abstract

The dynamic behavior of an endoglucanase from the hyperthermophilic microorganism Pyrococcus furiosus was investigated using elastic neutron scattering. The temperature dependence of the atomic motions was correlated with conformational and functional characteristics of the enzyme. The onset of biological function at temperatures higher than approximately 25 degrees C (the hyperthermostable enzyme is essentially inactive at room temperature) was associated with a dynamical transition in the anharmonic motions domain. This transition from the nonactive to the enzymatically active conformation involved structurally similar conformational substates in the energy landscape. From the mean-square displacement of the protein atoms, the molecular flexibility and the effective force constants were calculated at different temperature zones. The results showed that the activity increases at higher temperatures where the intramolecular bonds are weakened and the overall rigidity of the protein is decreased. Further temperature increase resulted in significantly increased atomic fluctuations featuring heat denaturation of the protein.

Explore related subjects

CalorimetryEnzyme StabilityGlucan Endo-1,3-beta-D-GlucosidaseMotionNeutronsProtein ConformationPyrococcus furiosusScattering, RadiationSpectrometry, FluorescenceTemperatureJournal ArticleResearch Support, Non-U.S. Gov't

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Sotirios Koutsopoulos, John van der Oost, Willem Norde. 2005-11-01. Temperature-dependent structural and functional features of a hyperthermostable enzyme using elastic neutron scattering.. https://doi.org/10.1002/prot.20606

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2007

Molecular determinants for the recruitment of the ubiquitin-ligase MuRF-1 onto M-line titin.

Titin forms an intrasarcomeric filament system in vertebrate striated muscle, which has elastic and signaling properties and is thereby central to mechanotransduction. Near its C-terminus and directly preceding a kinase domain, titin contains a conserved pattern of Ig and FnIII modules (Ig(A168)-Ig(A169)-FnIII(A170), hereby A168-A170) that recruits the E3 ubiquitin-ligase MuRF-1 to the filament. This interaction is thought to regulate myofibril turnover and the trophic state of muscle. We have elucidated the crystal structure of A168-A170, characterized MuRF-1 variants by circular dichroism (CD) and SEC-MALS, and studied the interaction of both components by isothermal calorimetry, SPOTS blots, and pull-down assays. This has led to the identification of the molecular determinants of the binding. A168-A170 shows an extended, rigid architecture, which is characterized by a shallow surface groove that spans its full length and a distinct loop protrusion in its middle point. In MuRF-1, a C-terminal helical domain is sufficient to bind A168-A170 with high affinity. This helical region predictably docks into the surface groove of A168-A170. Furthermore, pull-down assays demonstrate that the loop protrusion in A168-A170 is a key mediator of MuRF-1 recognition. Our findings indicate that this region of titin could serve as a target to attempt therapeutic inhibition of MuRF-1-mediated muscle turnover, where binding of small molecules to its distinctive structural features could block MuRF-1 access.

Calorimetry↗
PubMed2006

How does dextran sulfate prevent heat induced aggregation of protein? The mechanism and its limitation as aggregation inhibitor.

The effect of dextran sulfate on protein aggregation was investigated to provide the clues of its biochemical mechanism. The interaction between dextran sulfate and BSA varied with the pH values of the solution, which led to the different extent of aggregation prevention by dextran sulfate. Light scattering data with thermal scan showed that dextran sulfate suppressed BSA aggregation at pH 5.1 and pH 6.2, while it had no effect at pH 7.5. Isothermal titration calorimetric analysis suggested that the pH dependency of the role of dextran sulfate on BSA aggregation would be related to the difference in the mode of BSA-dextran sulfate complex formation. Isothermal titration calorimetric analysis at pH 6.2 indicated that dextran sulfate did not bind to native BSA at this pH, but interacted with partially unfolded BSA. While stabilizing native form of protein by the complex formation has been suggested as the suitable mechanism of preventing aggregation, our observation of conformational changes by circular dichroism spectroscopy showed that strong electrostatic interaction between dextran sulfate and BSA rather facilitated the denaturation of BSA. Combining the data from isothermal titration calorimetry, circular dichroism, and dynamic light scattering, we found that the complex formation of the intermediate state of denatured BSA with dextran sulfate is a prerequisite to suppress the aggregation by preventing further oligomerization/aggregation process of denatured protein.

Calorimetry↗
PubMed2006

Formulation, lyophilization and solid-state properties of a pegylated protein.

In this paper the importance of formulation and process parameters on the solid-state properties of a lyophilized, pegylated growth hormone antagonist (pegvisomant) was studied. The degree of solid-state disorder (amorphicity), protein/polyethylene glycol (PEG)/sucrose interactions, and dissolution characteristics of the resultant cakes were examined. Using isothermal microcalorimetry (IMC) and differential scanning calorimetry (DSC), it was shown that in co-lyophilized pegylated protein/sucrose systems there was an interaction between sucrose and pegylated protein molecules. This interaction was evidenced by a decrease in the melting temperature (Tm) and melting enthalpy of PEG as a function of sucrose concentration. It was also shown that the sum of the heat of interaction with water for the individual constituents, lyophilized pegylated protein and lyophilized sucrose, was higher than the heat of interaction for the co-lyophilized system. As the concentration of sucrose was increased, the degree of solid-state disorder increased and the solid dissolved faster. A correlation was found among heat of interaction with water, degree of solid-state disorder, and dissolution time. Pegylation caused a shorter dissolution time, lower moisture content, increased amorphicity, and a more rapid moisture-induced crystallization of sucrose.

Calorimetry↗