Ligand-Free Open-Closed Transitions of Periplasmic Binding Proteins: The Case of Glutamine-Binding Protein

Laboratory of Molecular Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Biochemistry (Impact Factor: 3.02). 02/2010; 49(9):1893-902. DOI: 10.1021/bi902045p
Source: PubMed


The ability to undergo large-scale domain rearrangements is essential for the substrate-binding function of periplasmic binding proteins (PBPs), which are indispensable for nutrient uptake in Gram-negative bacteria. Crystal structures indicate that PBPs typically adopt either an "open" unliganded configuration or a "closed" liganded one. However, it is not clear whether, as a general rule, PBPs remain open until ligand-induced interdomain closure or are in equilibrium with a minor population of unliganded, closed species. Evidence for the latter has been recently reported on maltose-binding protein (MBP) in aqueous solution [Tang, C., et al. (2007) Nature 449, 1078-1082] via paramagnetic relaxation enhancement (PRE), a technique able to probe lowly populated regions of conformational space. Here, we use PRE to study the unliganded open-closed transition of another PBP: glutamine-binding protein (GlnBP). Through a combination of domain structure knowledge and intermolecular and concentration dependence PRE experiments, a set of surface residues was found to be involved in intermolecular interactions. Barring such residues, PRE data on ligand-free GlnBP, paramagnetically labeled at two sites (one at a time), could be appropriately explained by the unliganded, open crystal structure in that it both yielded a good PRE fit and was not significantly affected by PRE-based refinement. Thus, contrary to MBP, our data did not particularly suggest the coexistence of a minor closed conformer. Several possibilities were explored to explain the observed differences in such closely structurally related systems; among them, a particularly interesting one arises from close inspection of the interdomain "hinge" region of various PBPs: strong hydrogen bond interactions discourage large-scale interdomain dynamics.

22 Reads
  • Source
    • "These measurements indicated that MBP could sample the HOLO state without ligand. This, however, might not be a general rule for all periplasmic binding proteins (Bermejo et al., 2010). Like NMR, the steep distance-dependence of FRET should allow for the identification of minor populations of proteins. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Mapping the landscape of a protein's conformational space is essential to understanding its functions and regulation. The limitations of many structural methods have made this process challenging for most proteins. Here, we report that transition metal ion FRET (tmFRET) can be used in a rapid, highly parallel screen, to determine distances from multiple locations within a protein at extremely low concentrations. The distances generated through this screen for the protein maltose binding protein (MBP) match distances from the crystal structure to within a few angstroms. Furthermore, energy transfer accurately detects structural changes during ligand binding. Finally, fluorescence-derived distances can be used to guide molecular simulations to find low energy states. Our results open the door to rapid, accurate mapping and prediction of protein structures at low concentrations, in large complex systems, and in living cells.
    Structure 12/2012; 21(1). DOI:10.1016/j.str.2012.11.013 · 5.62 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Author Summary A central goal of biology is to understand the function of the organism and its constituent parts at each of its scales of complexity. Function at the molecular level is often realized by changes in conformation. Unfortunately, experimental explorations of global motions critical for functional conformational changes are still challenging. In the present work, we developed a coarse grained triple-well structure-based model to explore the underlying functional landscape of maltose-binding protein (MBP). By quantitative flux analysis, we uncover the underlying mechanism by which the major induced fit and minor population shift pathways co-exist. Though we have previously lent credence to the assertion that dynamical equilibrium between open and minor closed conformations exist for all the free PBPs, the generality of this rule is still a matter of open debate. We found that the hinge flexibility is favorable to population shift mechanism. This finding provides a theoretica
  • [Show abstract] [Hide abstract]
    ABSTRACT: Several tools are already available for measuring IP traffic flows. However, a standard for exporting results is still missing and the IETF IPFIX WG (IP flow information export working group) is currently working towards this target. We present a prototype implementation of an IPFIX compliant flow meter and discuss its major challenges. Through an extensive profiling activity, we try to point out which operations are most critical for scalability issues. The latter can be raised by both a large number of contemporary flows and a high packet input rate. We finally provide some indicative figures of the incoming traffic rate, which can be reliably monitored with the platform we used to test our implementation.
    IP Operations and Management, 2003. (IPOM 2003). 3rd IEEE Workshop on; 11/2003
Show more


22 Reads
Available from