Complementing structural information of modular proteins with small angle neutron scattering and contrast variation.

Many macromolecules in the cell function by forming multi-component assemblies. We have applied the technique of small angle neutron scattering to study a nucleic acid-protein complex and a multi-protein complex. The results illustrate the versatility and applicability of the method to study macromolecular assemblies. The neutron scattering experiments, complementing X-ray solution scattering data, reveal that the conserved catalytic domain of RNase E, an essential ribonuclease in Escherichia coli (E. coli), undergoes a marked conformational change upon binding a 5'monophosphate-RNA substrate analogue. This provides the first evidence in support of an allosteric mechanism that brings about RNA substrate cleavage. Neutron contrast variation of the multi-protein TIM10 complex, a mitochondrial chaperone assembly comprising the subunits Tim9 and Tim10, has been used to determine a low-resolution shape reconstruction of the complex, highlighting the integral subunit organization. It shows characteristic features involving protrusions that could be assigned to the six subunits forming the complex.

Global conformational changes control the reactivity of methane monooxygenase.

We present here X-ray scattering data that yield new structural information on the multicomponent enzyme methane monooxygenase and its components: a hydroxylase dimer, and two copies each of a reductase and regulatory protein B. Upon formation of the enzyme complex, the hydroxylase undergoes a dramatic conformational change that is observed in the scattering data as a fundamental change in shape of the scattering particle such that one dimension is narrowed (by 25% or 24 A) while the longest dimension increases (by 20% or 25 A). These changes also are reflected in a 13% increase in radius of gyration upon complex formation. Both the reductase and protein B are required for inducing the conformational change. We have modeled the scattering data for the complex by systematically modifying the crystal structure of the hydroxylase and using ellipsoids to represent the reductase and protein B components. Our model indicates that protein B plays a role in optimizing the interaction between the active centers of the reductase and hydroxylase components, thus, facilitating electron transfer between them. In addition, the model suggests reasons why the hydroxylase exists as a dimer and that a possible role for the outlying gamma-subunit may be to stabilize the complex through its interaction with the other components. We further show that proteolysis of protein B to form the inactive B' results in a conformational change and B' does not bind to the hydroxylase. The truncation thus could represent a regulatory mechanism for controlling the enzyme activity.