Feldmanlauesen0539

Gd(III) complexes are currently established as spin labels for structural studies of biomolecules using pulse dipolar electron paramagnetic resonance (PD-EPR) techniques. This has been achieved by the availability of medium- and high-field spectrometers, understanding the spin physics underlying the spectroscopic properties of high spin Gd(III) (S=7/2) pairs and their dipolar interaction, the design of well-defined model compounds and optimization of measurement techniques. In addition, a variety of Gd(III) chelates and labeling schemes have allowed a broad scope of applications. In this review, we provide a brief background of the spectroscopic properties of Gd(III) pertinent for effective PD-EPR measurements and focus on the various labels available to date. We report on their use in PD-EPR applications and highlight their pros and cons for particular applications. We also devote a section to recent in-cell structural studies of proteins using Gd(III), which is an exciting new direction for Gd(III) spin labeling.The recent discoveries of the first proteins that bind lanthanides as part of their biological function not only are relevant to the emerging field of lanthanide-dependent biology, but also hold promise to revolutionize the technologically critical rare earths industry. Although protocols to assess the thermodynamics of metal-protein interactions are well established for "traditional" metal ions in biology, the characterization of lanthanide-binding proteins presents a challenge to biochemists due to the lanthanides' Lewis acidity, propensity for hydrolysis, and high-affinity complexes with biological ligands. These properties necessitate the preparation of metal stock solutions with very low buffered "free" metal concentrations (e.g., femtomolar to nanomolar) for such determinations. Herein we describe several protocols to overcome these challenges. First, we present standardization methods for the preparation of chelator-buffered solutions of lanthanide ions with easily calculated free metal concentrations. We also describe how these solutions can be used in concert with analytical methods including UV-visible spectrophotometry, circular dichroism spectroscopy, Förster resonance energy transfer (FRET), and sensitized terbium luminescence, in order to accurately determine dissociation constants (Kds) of lanthanide-protein complexes. Finally, we highlight how application of these methods to lanthanide-binding proteins, such as lanmodulin, has yielded insights into selective recognition of lanthanides in biology. We anticipate that these protocols will facilitate discovery and characterization of additional native lanthanide-binding proteins, will motivate the understanding of their biological context, and will prompt their applications in biotechnology.The chemical and physical properties of lanthanide coordination complexes can significantly change with small variations in their molecular structure. Further, in solution, coordination structures (e.g., lanthanide-ligand complexes) are dynamic. Resolving solution structures, computationally or experimentally, is challenging because structures in solution have limited spatial restrictions and are responsive to chemical or physical changes in their surroundings. To determine structures of lanthanide-ligand complexes in solution, a molecular simulation approach is presented in this chapter, which concurrently considers chemical reactions and molecular dynamics. Lanthanide ion, ligand, solvent, and anion molecules are explicitly included to identify, in atomic resolution, lanthanide coordination structures in solution. The computational protocol described is applicable to determining the molecular structure of lanthanide-ligand complexes, particularly with ligands known to bind lanthanides but whose structures have not been resolved, as well as with ligands not previously known to bind lanthanide ions. The approach in this chapter is also relevant to elucidating lanthanide coordination in more intricate structures, such as in the active site of enzymes.Infrared (IR) spectroscopy is a well-established technique for probing the structure, behavior, and surroundings of molecules in their native environments. Its characteristics-most specifically high structural sensitivity, ready applicability to aqueous samples, and broad availability-make it a valuable enzymological technique, particularly for the interrogation of ion binding sites. While IR spectroscopy of the "garden variety" (steady state at room temperature with wild-type proteins) is versatile and powerful in its own right, the combination of IR spectroscopy with specialized experimental schemes for leveraging ultrafast time resolution, protein labeling, and other enhancements further extends this utility. This book chapter provides the fundamental physical background and literature context essential for harnessing IR spectroscopy in the general context of enzymology with specific focus on interrogation of ion binding. Bafilomycin A1 chemical structure Studies of lanthanide ions binding to calmodulin are highlighted as illustrative examples of this process. Appropriate sample preparation, data collection, and spectral interpretation are discussed from a detail-oriented and practical perspective with the goal of facilitating the reader's rapid progression from reading words in a book to collecting and analyzing their own data in the lab.Single crystal X-ray diffraction is a technique that measures interatomic distances with atomic resolution. Utilizing this technique for metal complexes featuring lanthanide and actinide elements is complicated by the scarcity and radioactivity of many of the metals of the f-block, as sub-milligram samples are difficult to crystallize for small molecule X-ray diffraction experiments. In this chapter, we present a protocol developed in our group that circumvents these challenges by exploiting macromolecular crystallography, wherein a protein with a large and well-characterized binding calyx is used as a scaffold to crystallize small-molecule metal complexes. Highlighting several examples, we identify the structural and chemical information that can be acquired by this method, and delineate the benefits of directing crystal growth with proteins, such as decreasing the amount of metal used to the sub-microgram scale. Moreover, since protein recognition depends on the nature of the metal-chelator bonds, subtle effects in the lanthanide and actinide coordination chemistry, such as metal-ligand covalency, can be qualitatively assessed.