Molecular imaging holds considerable promise for elucidating biological processes in normal physiology as well as disease states, but requires noninvasive methods for identifying analytes at sub-micromolar concentrations. Particularly useful are genetically encoded, single-protein reporters that harness the power of molecular biology to visualize specific molecular processes, but such reporters have been conspicuously lacking for in vivo magnetic resonance imaging (MRI). Here, we report TEM-1 β-lactamase (bla) as a single-protein reporter for hyperpolarized (HP) 129Xe NMR, with significant saturation contrast at 0.1 μM. Xenon chemical exchange saturation transfer (CEST) interactions with the primary allosteric site in bla give rise to a unique saturation peak at 255 ppm, well removed (~60 ppm downfield) from the 129Xe-H2O peak. Useful saturation contrast was also observed for bla expressed in bacterial cells and mammalian cells.

Cryptophane-based biosensors are promising agents for the ultrasensitive detection of biomedically relevant targets via Xe NMR. Dynamic light scattering revealed that cryptophanes form water-soluble aggregates tens to hundreds of nanometers in size. Acridine orange fluorescence quenching assays allowed quantitation of the aggregation state, with critical concentrations ranging from 200 nM to 600 nM, depending on the cryptophane species in solution. The addition of excess carbonic anhydrase (CA) protein target to a benzenesulfonamide-functionalized cryptophane biosensor (C8B) led to C8B disaggregation and produced the expected 1:1 C8B-CA complex. C8B showed higher affinity at 298 K for the cytoplasmic isozyme CAII than the extracellular CAXII isozyme, which is a biomarker of cancer. Using hyper-CEST NMR, we explored the role of stoichiometry in detecting these two isozymes. Under CA-saturating conditions, we observed that isozyme CAII produces a largerXe NMR chemical shift change (δ = 5.9 ppm, relative to free biosensor) than CAXII (δ = 2.7 ppm), which indicates the strong potential for isozyme-specific detection. However, stoichiometry-dependent chemical shift data indicated that biosensor disaggregation contributes to the observed Xe NMR chemical shift change that is normally assigned to biosensor-target binding. Finally, we determined that monomeric cryptophane solutions improve hyper-CEST saturation contrast, which enables ultrasensitive detection of biosensor-protein complexes. These insights into cryptophane-solution behavior support further development of xenon biosensors, but will require reinterpretation of the data previously obtained for many water-soluble cryptophanes.

Protein cage self-assembly enables encapsulation and sequestration of small molecules, macromolecules, and nanomaterials for many applications in bionanotechnology. Notably, wild-type thermophilic ferritin from Archaeoglobus fulgidus (AfFtn) exists as a stable dimer of four-helix bundle proteins at low ionic strength, and the protein forms a hollow assembly of 24 protomers at high ionic strength (∼800 mM NaCl). This assembly process can also be initiated by highly charged gold nanoparticles (AuNPs) in solution, leading to encapsulation. These data suggest that salt solutions or charged AuNPs can shield unfavorable electrostatic interactions at AfFtn dimer-dimer interfaces, but specific “hot-spot” residues controlling assembly have not been identified. To investigate this further, we computationally designed three AfFtn mutants (E65R, D138K, A127R) that introduce a single positive charge at sites along the dimer-dimer interface. These proteins exhibited different assembly kinetics and thermodynamics, which were ranked in order of increasing 24mer propensity: A127R < WT < D138K ≪ E65R. E65R assembled to the 24mer across a wide range of ionic strengths (0 – 800 mM NaCl), and the dissociation temperature for the 24mer was 98 °C. X-ray crystal structure analysis of the E65R mutant identified a more compact, closed-pore cage geometry. A127R and D138K mutants exhibited wild-type ability to encapsulate and stabilize 5-nm AuNPs, whereas E65R gained ability to remain assembled in apo-form. This work illustrates designed protein cages with distinct assembly and encapsulation properties.

Genetically encoded magnetic resonance imaging (MRI) contrast agents enable non-invasive detection of specific biomarkers in vivo.

Indole prenyltransferases catalyze the prenylation of Ltryptophan (L-Trp) and other indoles to produce a diverse set of natural products in bacteria, fungi, and plants, many of which possess useful biological properties. Among this family of enzymes, CymD from Salinispora arenicola catalyzes the reverse N1 prenylation of L-Trp, an unusual reaction given the poor nucleophilicity of the indole nitrogen. CymD utilizes dimethylallyl diphosphate (DMAPP) as the prenyl donor, catalyzing the dissociation of the diphosphate leaving group followed by nucleophilic attack of the indole nitrogen at the tertiary carbon of the dimethylallyl cation. To better understand the structural basis of selective indole N-alkylation reactions in biology, we have determined the X-ray crystal structures of CymD, the CymD−L-Trp complex, and the CymD−L-Trp−DMSPP complex (DMSPP is dimethylallyl S-thiolodiphosphate, an unreactive analogue of DMAPP). The orientation of L-Trp with respect to DMSPP reveals how the active site contour of CymD serves as a template to direct the reverse prenylation of the indole nitrogen. Comparison to PriB, a C6 bacterial indole prenyltransferase, offers further insight regarding the structural basis of regioselective indole prenylation. Isothermal titration calorimetry measurements indicate a synergistic relationship between L-Trp and DMSPP binding. Finally, activity assays demonstrate the selectivity of CymD for L-Trp and indole as prenyl acceptors. Collectively, these data establish a foundation for understanding and engineering the regioselectivity of indole prenylation by members of the prenyltransferase protein family.

Here, we report a supramolecular strategy for detecting specific proteins in complex media using hyperpolarized 129Xe NMR. A cucurbit[6]uril (CB[6]) based molecular relay was programmed for three sequential equilibrium conditions by designing a two-faced guest (TFG) that initially binds CB[6] and blocks CB[6]-Xe interaction. Protein analyte recruits the TFG and frees CB[6] for Xe binding. TFGs containing CB[6]- and carbonic anhydrase II (CAII)-binding domains were synthesized in one or two steps. X-ray crystallography confirmed TFG binding to Zn2+ in the deep, active-site CAII cleft, which precludes simultaneous CB[6] binding. The molecular relay was reprogrammed to detect avidin using a different TFG. Finally, CB[6]-Xe binding was detected in buffer and in E. coli cultures expressing CAII via ultrasensitive 129Xe NMR spectroscopy.

The regiospecific prenylation of an aromatic amino acid catalyzed by a dimethylallyl-L-tryptophan synthase (DMATS) is a key step in the biosynthesis of many fungal and bacterial natural products. DMATS enzymes share a common "ABBA" fold with divergent active site contours that direct alternative C−C, C−N, and C−O bond-forming trajectories. DMATS1 from Fusarium f ujikuroi catalyzes the reverse N-prenylation of L-Trp by generating an allylic carbocation from dimethylallyl diphosphate (DMAPP) that then alkylates the indole nitrogen of L-Trp. DMATS1 stands out among the greater DMATS family because it exhibits unusually broad substrate specificity: it can utilize geranyl diphosphate (GPP) or L-Tyr as an alternative prenyl donor or acceptor, respectively; it can catalyze both forward and reverse prenylation, i.e., at C1 or C3 of DMAPP; and it can catalyze C−N and C−O bond-forming reactions. Here, we report the crystal structures of DMATS1 and its complexes with L-Trp or L-Tyr and unreactive thiolodiphosphate analogues of the prenyl donors DMAPP and GPP. Structures of ternary complexes mimic Michaelis complexes with actual substrates and illuminate active site features that govern prenylation regiochemistry. Comparison with CymD, a bacterial enzyme that catalyzes the reverse N-prenylation of L-Trp with DMAPP, indicates that bacterial and fungal DMATS enzymes share a conserved reaction mechanism. However, the narrower active site contour of CymD enforces narrower substrate specificity. Structure−function relationships established for DMATS enzymes will ultimately inform protein engineering experiments that will broaden the utility of these enzymes as useful tools for synthetic biology.

The physiological activity of xenon has long been recognized, though the exact nature of its interactions with biomolecules remains poorly understood. Xe is an inert noble gas, but can act as a general anesthetic, most likely by binding internal hydrophobic cavities within proteins. Understanding Xe-protein interactions, therefore, can provide crucial insight regarding the mechanism of Xe anesthesia and potentially other general anesthetic agents. Historically, Xe-protein interactions have been studied primarily through X-ray crystallography and nuclear magnetic resonance (NMR). In this chapter, we first describe our methods for preparing Xe derivatives of protein crystals and identifying Xe-binding sites. Second, we detail our procedure for Xe hyper-CEST NMR spectroscopy, a versatile NMR technique well suited for characterizing the weak, transient nature of Xe-protein interactions.