Neutron Scattering Reveals the Dynamic Basis of Protein Adaptation to Extreme Temperature

Abstract

To explore protein adaptation to extremely high temperatures, two parameters related to macromolecular dynamics, the mean square atomic fluctuation and structural resilience, expressed as a mean force constant, were measured by neutron scattering for hyperthermophilic malate dehydrogenase from Methanococcus jannaschii and a mesophilic homologue, lactate dehydrogenase from Oryctolagus cunniculus (rabbit) muscle. The root mean square fluctuations, defining flexibility, were found to be similar for both enzymes (1.5 Å) at their optimal activity temperature. Resilience values, defining structural rigidity, are higher by an order of magnitude for the high temperature-adapted protein (0.15 Newtons/meter for O. cunniculus lactate dehydrogenase and 1.5 Newtons/meter for M. jannaschii malate dehydrogenase). Thermoadaptation appears to have been achieved by evolution through selection of appropriate structural rigidity in order to preserve specific protein structure while allowing the conformational flexibility required for activity.Hyperthermophilic organisms grow at temperatures above 80°C. Proteins from these organisms are themselves optimally active between 60 and 125°C and serve as paradigms for the characterization of factors responsible for protein fold stability and flexibility. Hyperthermophilic enzymes have also attracted considerable attention because of a range of biotechnological applications (1, 2).Sequence comparison studies and structural analyses carried out on hyperthermophilic proteins and their mesophilic homologues have shown that thermal stability is associated with multiple factors, including an increase in hydrogen bonding, complex salt bridge formation, and helix stabilization by acidic residues. The commonly accepted hypothesis is that increased thermal stability is due to enhanced conformational rigidity of the molecular structure (3). Hyperthermophilic enzymes are also characterized by a higher temperature of maximum activity (3, 4). The more rigid hyperthermophilic enzyme would then require higher temperatures to achieve the requisite conformational flexibility for activity.Experiments have shown that thermostable enzymes exhibit reduced structural flexibility at room temperature with respect to their mesophilic homologues (4, 5), whereas others, on ␣-amylase (6) and on rubredoxin (7,8), have shown the opposite effect, i.e. the thermostable homologues were found to be more flexible, suggesting stabilization through entropic effects. Relations between flexibility and stability are, therefore, complex. Atomic fluctuations only were measured in these experiments and interpreted in terms of flexibility.It is important to point out that reduced structural "flexibility" does not necessarily imply a more "rigid" structure. Atoms are maintained in a structure by forces that link them to their neighbors. In terms of a force field, the width of the potential well in which an atom moves is a measure of its flexibility in terms of a root mean square fluctuation amplitude ͑ ͌ Ͻu 2 Ͼ), whereas the detailed ...

Fundamental and biotechnological applications of neutron scattering measurements for macromolecular dynamics

“…The guanidinium chloride unfolding transition concentrations for Oc LDH and Mj MalDH are 1.3 and 2.1 M, respectively. Taken together, these data show that the hyperthermophilic protein is significantly more stable with a temperature of optimal activity higher (Tehei et al 2005). …”

“…Dynamics on the picosecond to nanosecond timescale allow macromolecules to achieve the stability and fluctuations, and, therefore, the necessary rigidity and flexibility to perform their biological functions (Lehnert et al 1998;Tehei et al 2001Tehei et al , 2005. Neutron spectroscopy is particularly adapted to the study of these motions, because neutron wavelengths (% Å ) and energies (% meV) match, respectively, the amplitudes and frequencies of molecular motions (Gabel et al 2002;Smith 1991).…”

“…Firstly, the enzyme malate dehydrogenase from the hyperthermophile Archaeon Methanococcus jannaschii (Mj MalDH) (Tehei et al 2005). This extremophile, like all organisms living in extreme environments (extremophiles) has to develop molecular mechanisms of adaptation to one or several more extreme physicochemical conditions.…”

New insights into microbial adaptation to extreme saline environments

“…Adaptation of proteome molecular dynamics has been revealed by neutron scattering in bacteria living from different temperature environments (17). Adaptation to high temperature, for instace, does not modify in depth the overall tri-dimensional fold of proteins but proceeds instead by an increase in the intramolecular interactions, a reduction of the loops and a more hydrophobic core (18,19). Protein surface modifications such as acidification or enrichments in stable aminoacids also contribute to stabilize the macromolecule in extreme conditions.…”

“…Protein surface modifications such as acidification or enrichments in stable aminoacids also contribute to stabilize the macromolecule in extreme conditions. By using neutron spectrometry we have shown that these modifications suffice to modify drastically the molecular dynamic properties of a protein, leading to an increase in its mean resilience (18). The study of the enzymatic, biophysical and structural properties lactate-malate dehydrogenases arising from different organisms has shown that a few hot spots in the protein structure are selected by evolution to maintain an adequate local flexibility in order to permit its biological function (19).…”