An official website of the United States government
The biological importance of lanthanides, and the early lanthanides (La 3+ –Nd 3+ ) in particular, has only recently been recognized, and the structural principles underlying selective binding of lanthanide ions in biology are not yet well established. Lanmodulin (LanM) is a novel protein that displays unprecedented affinity and selectivity for lanthanides over most other metal ions, with an uncommon preference for the early lanthanides. Its utilization of EF-hand motifs to bind lanthanides, rather than the Ca 2+ typically recognized by these motifs in other proteins, has led it to be used as a model system to understand selective lanthanide recognition. Two-dimensional infrared (2D IR) spectroscopy combined with molecular dynamics simulations were used to investigate LanM's selectivity mechanisms by characterizing local binding site geometries upon coordination of early and late lanthanides as well as calcium. These studies focused on the protein's uniquely conserved proline residues in the second position of each EF-hand binding loop. We found that these prolines constrain the EF-hands for strong coordination of early lanthanides. Substitution of this proline results in a more flexible binding site to accommodate a larger range of ions but also results in less compact coordination geometries and greater disorder within the binding site. Finally, we identify the conserved glycine in the sixth position of each EF-hand as a mediator of local binding site conformation and global secondary structure. Uncovering fundamental structure–function relationships in LanM informs the development of synthetic biology technologies targeting lanthanides in industrial applications.
more »
« less
Chapter Six - Heterologous expression, purification, and characterization of proteins in the lanthanome
Recent work has revealed that certain lanthanides—in particular, the more earth-abundant, lighter lanthanides—play essential roles in pyrroloquinoline quinone (PQQ) dependent alcohol dehydrogenases from methylotrophic and non-methylotrophic bacteria. More recently, efforts of several laboratories have begun to identify the molecular players (the lanthanome) involved in selective uptake, recognition, and utilization of lanthanides within the cell. In this chapter, we present protocols for the heterologous expression in Escherichia coli, purification, and characterization of many of the currently known proteins that comprise the lanthanome of the model facultative methylotroph, Methylorubrum extorquens AM1. In addition to the methanol dehydrogenase XoxF, these proteins include the associated c-type cytochrome, XoxG, and solute binding protein, XoxJ. We also present new, streamlined protocols for purification of the highly selective lanthanide-binding protein, lanmodulin, and a solute binding protein for PQQ, PqqT. Finally, we discuss simple, spectroscopic methods for determining lanthanide- and PQQ-binding stoichiometry of proteins. We envision that these protocols will be useful to investigators identifying and characterizing novel members of the lanthanome in many organisms.
more »
« less
- Award ID(s):
- 1945015
- PAR ID:
- 10226154
- Editor(s):
- Cotruvo Jr, Joseph A
- Date Published:
- Journal Name:
- Methods in enzymology
- Volume:
- 650
- ISSN:
- 0076-6879
- Page Range / eLocation ID:
- 119-157
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Elucidating details of biology’s selective uptake and trafficking of rare earth elements, particularly the lanthanides, has the potential to inspire sustainable biomolecular separations of these essential metals for myriad modern technologies. Here, we biochemically and structurally characterizeMethylobacterium(Methylorubrum)extorquensLanD, a periplasmic protein from a bacterial gene cluster for lanthanide uptake. This protein provides only four ligands at its surface-exposed lanthanide-binding site, allowing for metal-centered protein dimerization that favors the largest lanthanide, LaIII. However, the monomer prefers NdIIIand SmIII, which are disfavored lanthanides for cellular utilization. Structure-guided mutagenesis of a metal-ligand and an outer-sphere residue weakens metal binding to the LanD monomer and enhances dimerization for PrIIIand NdIIIby 100-fold. Selective dimerization enriches high-value PrIIIand NdIIIrelative to low-value LaIIIand CeIIIin an all-aqueous process, achieving higher separation factors than lanmodulins and comparable or better separation factors than common industrial extractants. Finally, we show that LanD interacts with lanmodulin (LanM), a previously characterized periplasmic protein that shares LanD’s preference for NdIIIand SmIII. Our results suggest that LanD’s unusual metal-binding site transfers less-desirable lanthanides to LanM to siphon them away from the pathway for cytosolic import. The properties of LanD show how relatively weak chelators can achieve high selectivity, and they form the basis for the design of protein dimers for separation of adjacent lanthanide pairs and other metal ions.more » « less
-
Abstract Lanthanides, which are part of the rare earth elements group have numerous applications in electronics, medicine and energy storage. However, our ability to extract them is not meeting the rapidly increasing demand. The discovery of the bacterial periplasmic lanthanide‐binding protein lanmodulin spurred significant interest in developing biotechnological routes for lanthanide detection and extraction. Here we report the construction of β‐lactamase‐lanmodulin chimeras that function as lanthanide‐controlled enzymatic switches. Optimized switches demonstrated dynamic ranges approaching 3000‐fold and could accurately quantify lanthanide ions in simple colorimetric or electrochemical assays.E.colicells expressing such chimeras grow on β‐lactam antibiotics only in the presence of lanthanide ions. The developed lanthanide‐controlled protein switches represent a novel platform for engineering metal‐binding proteins for biosensing and microbial engineering.more » « less
-
Immunoaffinity chromatography (IAC) is a type of liquid chromatography that uses immobilized antibodies or related binding agents as selective stationary phases for sample separation or analysis. The strong binding and high selectivity of antibodies have made IAC a popular tool for the purification and analysis of many chemicals and biochemicals, including proteins. The basic principles of IAC are described as related to the use of this method for protein purification and analysis. The main factors to consider in this technique are also presented under a discussion of the general strategy to follow during the development of a new IAC method. Protocols, as illustrated using human serum albumin (HSA) as a model protein, are provided for the use of IAC in several formats. This includes both the use of IAC with traditional low‐performance supports such as agarose for off‐line immunoextraction and supports used in high‐performance IAC for on‐line immunoextraction. The use of IAC for protein analysis as a flow‐based or chromatographic immunoassay is also discussed and described using HSA and a competitive binding assay format as an example.more » « less
-
Lanthanides (Ln) are a new group of life metals, and many questions remain regarding how they are acquired and used in biology. Methylotrophic bacteria can acquire, transport, biomineralize, and use Ln as part of a cofactor complex with pyrroloquinoline quinone (PQQ) in alcohol dehydrogenases. For most methylotrophic bacteria use is restricted to the light Ln, which range from lanthanum to samarium (atomic numbers 57–62). Understanding how the cell differentiates between light and heavy Ln, and the impacts of these metals on the metabolic network, will advance the field of Ln biochemistry and give insights into enzyme catalysis, stress homeostasis, and metal biomineralization and compartmentalization. We report robust methanol growth with the heavy Ln gadolinium by a genetic variant of the model methylotrophic bacterium Methylorubrum extorquens AM1, named evo -HLn, for “ evo lved for H eavy L antha n ides.” A non-synonymous single nucleotide polymorphism in a cytosolic hybrid histidine kinase/response regulator allowed for sweeping transcriptional alterations to heavy metal stress response, methanol oxidation, and central metabolism. Increased expression of genes for Ln acquisition and uptake, production of the Ln-chelating lanthanophore, PQQ biosynthesis, and phosphate transport and metabolism resulted in gadolinium hyperaccumulation of 36-fold with a trade-off for light Ln accumulation. Gadolinium was hyperaccumulated in an enlarged acidocalcisome-like compartment. This is the first evidence of a bacterial intracellular Ln-containing compartment that we name the “lanthasome.” Carotenoid and toblerol biosynthesis were also upregulated. Due to its unique capabilities, evo -HLn can be used to further magnetic resonance imaging (MRI) and bioremediation technologies. In this regard, we show that gadolinium hyperaccumulation was sufficient to produce MRI contrast in whole cells, and that evo -HLn was able to readily acquire the metal from the MRI contrast agent gadopentetic acid. Finally, hyperaccumulation of gadolinium, differential uptake of light and heavy Ln, increased PQQ levels, and phosphate transport provide new insights into strategies for Ln recovery.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1016/bs.mie.2021.02.004
