Intellectual Merit: Lanthanide binding tags (LBTs) are small peptides (15-20 residues) that impart on the protein to which they are attached specific and tight binding of lanthanide ions. LBTs permit the use of applications that rely upon the photophysical properties of the bound lanthanides. These sequences have been integrated into many target proteins and tested for applications in nuclear magnetic resonance, X-ray crystallography, and luminescence. In the next phase of research, the LBT technology will be enlisted for addressing challenging problems of biological significance, moving from the test tube to the cell. The new studies will exploit the unique properties of the LBTs and the bound lanthanides. Namely, lanthanides luminescence properties can eliminate the high background often complicating measurements in biological systems using current technologies for locating proteins and measuring protein motion. The LBT capabilities will be extended by modification of the chemistry to impart properties with greater number of potential application. There are four parts to the project: 1) The LBTs will be used to study dynamic changes in protein structure using the property of luminescence resonance energy transfer in the important cell signaling calcineurin A/calcineurin B complex. 2) These new LBTs will be used to increase understanding of cell-cell signaling and cellular trafficking. 3) The LBT will be developed as a novel gadolinium contrast agent for use in magnetic resonance imaging. 4) The use of LBTs in determining protein crystal structures will be extended to the challenging problem of solution structure of proteins embedded in cell membranes. The LBTs will provide multiple features for the solution of these difficult structures. The dissemination of the information gained from these studies will allow the broad application of LBTs by the research communities. During the development of the tags, high school students, graduate students and postdoctoral associates will been trained in an integrated and collaborative research environment spanning multiple disciplines. These studies will provide the researchers with the foundation needed to address a broad range of problems at the interface of Biology, Chemistry and Physics. Broader Impact of the Research. During the project period, use of the LBTs will be extended in applications of biological importance, providing the scientific community with new LBTs and defined protocols for the application of the tags to a wide variety of problems including the study of protein dynamics, protein signaling and trafficking, cellular environment reporting, and the structure of integral membrane proteins. During the development of the tags, students and postdoctoral associates will be trained in an integrated and collaborative research environment spanning skill sets in molecular biology, fluorescence, protein chemistry, MRI, and X-ray crystallography at multiple institutions. These studies will provide the researchers with the foundation needed to address a broad range of problems at the interface of Biology, Chemistry and Physics. Undergraduate students will have the opportunity to be trained under the auspices of the UROP (undergraduate research opportunities) programs at MIT and Boston University School of Medicine as well as High School students through the BU CityLab program. Both MIT and Boston University have aggressive policies and procedures in action for the recruitment and retention of under-represented minorities at the undergraduate and graduate levels.
Lanthanide Binding Tags: The Swiss Army Knife of Protein Tags! MRI is a central tool in medical diagnostics, enabling non-invasive visualization of soft-tissues. Targeted MRI approaches utilize complexes of the lanthanide ion gadolinium that are attached to homing molecules that bind to disease-specific proteins. Through rational design using three-dimensional structure analysis we developed a peptide-based lanthanide-binding tag (LBT) that complexes gadolinium tightly and allows visualization with high contrast. The LBT can be easily appended to proteins using standard molecular biology methods, which allows facile incorporation of the LBT-MRI tag to any proteins of interest. This modular nature of LBTs and ability to generate large collections of LBTs based on the prototype LBT-MRI that we have built and tested sets the stage for a new generation of molecular imaging agents. Membrane proteins (MPs) are protein molecules that are found tightly associated with the membrane bilayers of all cells and account for about a third of the proteins found in the human body. MPs perform very important functions. For example, some MPs form channels or pumps to transport cargo in and out of cells and others act as receptors that bind molecules outside the cell and translate these signals into cellular activities. MPs are critical for the proper function of all living systems and they are also frequently implicated in human diseases. Therefore, they represent a huge proportion (more than 60%) of existing and under development drug targets. For example, G-protein-coupled receptors (GPCRs) form a large and vital family of drug targets among membrane proteins because the malfunction of these receptors results in serious disorders, such as hypertension, congestive heart failure, stroke, and cancer. MPs have proven to be very difficult to study because their structures are often disrupted when they are removed from the membrane location where they are normally found and therefore, there is an urgent need for new tools to help to study the structures and functions of membrane proteins. Our NSF funded research on lanthanide binding tags (LBTs) has provided an important new multifunctional tool for studying membrane proteins. Since the LBTs are short protein sequences they can be built into the structures of membrane proteins without compromising normal function. Our research has shown that LBTs can be readily inserted into existing loops in the protein structure or added to ends of the sequences. Because of the chemical nature of lanthanides, the lanthanide ion bound to the LBT can be used to visualize the tagged protein and give valuable information about the location in the cell, the interactions between proteins and other cellular components, and the changes of protein structure that accompany activity. The LBTs are ideally suited to study the multitude of protein targets for the purpose of drug discovery and diagnostics.