Grantee Research Project Results
Final Report: NMR Structural Studies of Mercury Transport Proteins
EPA Grant Number: R823576Title: NMR Structural Studies of Mercury Transport Proteins
Investigators: Opella, Stanley J.
Institution: University of Pennsylvania
EPA Project Officer: Hahn, Intaek
Project Period: October 1, 1995 through September 1, 1998
Project Amount: $404,667
RFA: Exploratory Research - Environmental Biology (1995) RFA Text | Recipients Lists
Research Category: Biology/Life Sciences , Human Health , Aquatic Ecosystems
Objective:
The overall goal of the project was to initiate and evaluate the application of the methods of structural biology to an environmental problem, in particular bioremediation of heavy metal toxins. The specific goals were associated with NMR structural studies of two proteins, merP and merT, of the bacterial mercury detoxification system. They included the development of expressions systems, isotopic labeling, purification, and then performing NMR studies to determine their structures and describe their dynamics. The information gained from these studies provides insight into the fundamental mechanisms of binding and transport of heavy metal toxins.
Summary/Accomplishments (Outputs/Outcomes):
The two proteins investigated from the bacterial mercury detoxification system are responsible for the binding of Hg(II) in the cell periplasm (merP) and for transporting Hg(II) across the cell membrane into the cell (merT) have quite different properties. MerP is a water soluble, globular protein and merT is a hydrophobic membrane protein. Therefore, these proteins required quite different NMR approaches for structure determination. Multidimensional solution NMR methods worked well for merP in aqueous solution and we were able to determine the structures of both the metal-free and metal-bound forms of the protein to high resolution. Membrane proteins like merT require a much more sophisticated and complex approach to structure determination. MerT was studied in parallel by solution NMR methods in micelle samples and by solid-state NMR methods in bilayers samples, both of which required substantial methodological development. Substantial progress was made toward the characterization of the structure of merT, including delineation of its secondary structure and preliminary results indicating the overall fold of the proteins. We were able to formulate a model of the protein based on experimental results for comparison with previous models based on sequence analysis and biological properties.
This research has the potential to contribute to the improvement in risk assessment through the development of protein based sensors and to the improvement in risk management through the development of protein based devices for bioremediation.
MerP: the periplasmic mercury binding protein
MerP is a water soluble, globular protein. Its primary role appears to be that of a scavenger of free Hg(II) in the cell periplasm, presumably to protect proteins associated with outside of the cell from Hg(II) containing toxins. After binding Hg(II), merP passes the metal ions to the next protein in the chain, the membrane transport protein merT. The metal ion is then transported across the cell membrane into the cytoplasm by the action of merT. Hg(II) is reduced by the enzyme mercuric reductase, merA, to Hg(O) in the cytoplasm.
The principal findings of the research supported by the EPA grant are the three-dimensional structures of reduced (metal-free) and mercury-bound forms of merP in aqueous solution by NMR spectroscopy. These structures are shown in Figure 1. Both forms of the protein have essentially the same global fold, which consists of two antiparallel alpha helices overlaying a four strand beta sheet. The major differences between the two forms of merP, which include both spectroscopic properties (chemical shifts, relaxation parameters) and backbone and sidechain structures, are localized in residues 10 - 18, which constitute the loop that binds Hg(II). This is an extremely important region of the protein. It contains the highly conserved residues, GMTCAAC, which includes the two cysteine residues that contribute the sidechain -SH groups for metal ligation. Direct comparisons of the structures show that binding of the mercury causes a slight unwinding of the helix as the two Cys residues become closer. Another difference is in the position of the aromatic residue phenylalanine 38, which lies below the metal binding loop. In the reduced form, this sidechain is oriented towards the binding loop; in contrast the aromatic ring moves closer to the surface of the protein in the mercury-bound form. This may have implications for how the protein interacts with the membrane or the membrane associated protein merT.
Figure 1: Structures of merP determined by multidimensional solution NMR spectroscopy. A. and B. are superpositions of the 20 lowest energy structures calculated from the NMR data. A. Reduced (metal-free) merP. B. Mercury-bound merP. C. and D. are MOLSCRIPT representations of the structures of merP. C. Reduced (metal-free) merP. D. Mercury-bound merP.
MerT, the membrane protein that transports mercury
Both micelle and bilayer samples of merT were prepared for NMR experiments. The samples prepared for the various NMR experiments represent the full range of isotopic labeling for NMR spectroscopy. This includes uniform 15N; uniform 13C and 15N; uniform 2H and 15N; uniform 2H, 13C, and 15N; and selective 15N. The successful preparation of these samples reflected the intensive effort put into the molecular biology and preparative biochemistry of small membrane proteins by the research program, in particular for the studies of merT. We have found it essential to express hydrophobic membrane proteins as fusion proteins, relying on the properties of the fusion partner to keep the hydrophobic membrane protein away from the cell membrane.
We prepared a fusion of maltose binding protein and merT. Mass spectrometry, amino acid analysis, and limited N-terminal sequencing demonstrated that the polypeptide ultimately purified from the expression corresponds to the correct protein. Both micelle and bilayer samples of isotopically labeled merT were prepared from this expression system. The results from the solution NMR experiments enabled us to characterize the secondary structure of the protein and those from the solid-state NMR experiments gave some information about the organization of the protein in bilayers. The current model for merT based on our experimental NMR results is shown in Figure 2.
Figure 2: Model of merT in membrane bilayers based on experimental NMR results.
Conclusions:
The immediate focus of the structural biology is on the protein than binds mercury (merP) and the protein that transports mercury across the membrane (merT). Even at this early stage of our investigations, structural results that shed light on the functions of merP and merT are available. Just the ability to pose specific questions about chemical interactions and structural changes represents a major advance in understanding this system, in particular, and the initial application of structural biology to an environmental problem, in general.
The three-dimensional structures of merP and merT, in combination with the ongoing structural studies of other structural proteins of the mer operon in other laboratories will provide considerable insight into the chemistry and structural biology of the bacterial mercury detoxification system. Once the structures of these proteins are completely determined, it may be possible to re-engineer them through various site-directed and random mutagenesis methods to optimize their functions, alter their metal binding specificity, and stabilize their structures. It may then be feasible to place the altered protein in natural or synthetic membranes to make biologically based devices for the detection and separation of mercury and other metals.
Journal Articles on this Report : 1 Displayed | Download in RIS Format
Other project views: | All 2 publications | 1 publications in selected types | All 1 journal articles |
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Steele RA, Opella SJ. Structures of the reduced and mercury-bound forms of MerP, the periplasmic protein from the bacterial mercury detoxification system. Biochemistry 1997;36(23):6885-6895. |
R823576 (Final) |
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Supplemental Keywords:
RFA, Scientific Discipline, Water, Environmental Chemistry, Biochemistry, Bioremediation, Biology, Mercury, volatile metallic mercury , fate and transport, bacteria control, biodegradation, chemical transport, contaminant release, mercury transport proteins, volatile metallic mercury, NMR spectroscopy , mercury concentration, bacterial degradation, heavy metalsProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.