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1.
Biochemistry ; 58(36): 3755-3766, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31436969

RESUMO

Polyamines are small organic cations that are essential for cellular function in all kingdoms of life. Polyamine metabolism is regulated by enzyme-catalyzed acetylation-deacetylation cycles in a fashion similar to the epigenetic regulation of histone function in eukaryotes. Bacterial polyamine deacetylases are particularly intriguing, because these enzymes share the fold and function of eukaryotic histone deacetylases. Recently, acetylpolyamine amidohydrolase from the deep earth halophile Marinobacter subterrani (msAPAH) was described. This Zn2+-dependent deacetylase shares 53% amino acid sequence identity with the acetylpolyamine amidohydrolase from Mycoplana ramosa (mrAPAH) and 22% amino acid sequence identity with the catalytic domain of histone deacetylase 10 from Danio rerio (zebrafish; zHDAC10), the eukaryotic polyamine deacetylase. The X-ray crystal structure of msAPAH, determined in complexes with seven different inhibitors as well as the acetate coproduct, shows how the chemical strategy of Zn2+-dependent amide hydrolysis and the catalytic specificity for cationic polyamine substrates is conserved in a subterranean halophile. Structural comparisons with mrAPAH reveal that an array of aspartate and glutamate residues unique to msAPAH enable the binding of one or more Mg2+ ions in the active site and elsewhere on the protein surface. Notwithstanding these differences, activity assays with a panel of acetylpolyamine and acetyllysine substrates confirm that msAPAH is a broad-specificity polyamine deacetylase, much like mrAPAH. The broad substrate specificity contrasts with the narrow substrate specificity of zHDAC10, which is highly specific for N8-acetylspermidine hydrolysis. Notably, quaternary structural features govern the substrate specificity of msAPAH and mrAPAH, whereas tertiary structural features govern the substrate specificity of zHDAC10.

2.
Biochemistry ; 58(30): 3232-3242, 2019 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-31251043

RESUMO

Indole prenyltransferases catalyze the prenylation of l-tryptophan (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.

3.
Chemphyschem ; 20(2): 260-267, 2019 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-30151973

RESUMO

Genetically encoded (GE) contrast agents detectable by magnetic resonance imaging (MRI) enable non-invasive visualization of gene expression and cell proliferation at virtually unlimited penetration depths. Using hyperpolarized 129 Xe in combination with chemical exchange saturation transfer, an MR contrast approach known as hyper-CEST, enables ultrasensitive protein detection and biomolecular imaging. GE MRI contrast agents developed to date include nanoscale proteinaceous gas vesicles as well as the monomeric bacterial proteins TEM-1 ß-lactamase (bla) and maltose binding protein (MBP). To improve understanding of hyper-CEST NMR with proteins, structural and computational studies were performed to further characterize the Xe-bla interaction. X-ray crystallography validated the location of a high-occupancy Xe binding site predicted by MD simulations, and mutagenesis experiments confirmed this Xe site as the origin of the observed CEST contrast. Structural studies and MD simulations with representative bla mutants offered additional insight regarding the relationship between local protein structure and CEST contrast.


Assuntos
Isótopos de Xenônio/química , beta-Lactamases/química , Sítio Alostérico , Sítios de Ligação , Meios de Contraste/química , Cristalografia por Raios X , Limite de Detecção , Proteínas Ligantes de Maltose/química , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica
4.
Anal Chem ; 90(12): 7730-7738, 2018 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-29782149

RESUMO

Cryptophane-based biosensors are promising agents for the ultrasensitive detection of biomedically relevant targets via 129Xe 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 larger 129Xe 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 129Xe 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.


Assuntos
Técnicas Biossensoriais , Anidrases Carbônicas/análise , Técnicas Eletroquímicas , Nanoestruturas/química , Ressonância Magnética Nuclear Biomolecular , Compostos Policíclicos/química , Técnicas Biossensoriais/instrumentação , Anidrases Carbônicas/isolamento & purificação , Anidrases Carbônicas/metabolismo , Técnicas Eletroquímicas/instrumentação , Fluorescência , Humanos , Solubilidade , Isótopos de Xenônio
5.
Methods Enzymol ; 602: 249-272, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29588032

RESUMO

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 129Xe hyper-CEST NMR spectroscopy, a versatile NMR technique well suited for characterizing the weak, transient nature of Xe-protein interactions.


Assuntos
Anestésicos Inalatórios/farmacologia , Mioglobina/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , Isótopos de Xenônio/farmacologia , Anestésicos Inalatórios/química , Sítios de Ligação , Cristalografia por Raios X , Interações Hidrofóbicas e Hidrofílicas , Mioglobina/química , Isótopos de Xenônio/química
6.
Biochemistry ; 56(28): 3596-3606, 2017 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-28682599

RESUMO

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 a low ionic strength, and the protein forms a hollow assembly of 24 protomers at a 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, and 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 < wild type < D138K ≪ E65R. E65R assembled into 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 did not encapsulate AuNPs at the same high yields. This work illustrates designed protein cages with distinct assembly and encapsulation properties.


Assuntos
Proteínas Arqueais/química , Archaeoglobus fulgidus/química , Ferritinas/química , Proteínas Arqueais/genética , Archaeoglobus fulgidus/genética , Cristalografia por Raios X , Composição de Medicamentos , Ferritinas/genética , Ouro/química , Nanopartículas Metálicas/química , Modelos Moleculares , Mutação Puntual , Multimerização Proteica , Eletricidade Estática , Termodinâmica
8.
Angew Chem Int Ed Engl ; 55(31): 8984-7, 2016 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-27305488

RESUMO

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). Herein, we report TEM-1 ß-lactamase (bla) as a single-protein reporter for hyperpolarized (HP) (129) Xe 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 (129) Xe-H2 O peak. Useful saturation contrast was also observed for bla expressed in bacterial cells and mammalian cells.


Assuntos
Imagem Molecular , Xenônio/metabolismo , beta-Lactamases/metabolismo , Células HEK293 , Humanos , Espectroscopia de Ressonância Magnética , Xenônio/química , Isótopos de Xenônio , beta-Lactamases/química
9.
Angew Chem Int Ed Engl ; 55(5): 1733-6, 2016 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-26692420

RESUMO

A supramolecular strategy for detecting specific proteins in complex media by using hyperpolarized (129) Xe NMR is reported. 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 the CB[6]-Xe interaction. The 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 Zn(2+) in the deep CAII active-site cleft, which precludes simultaneous CB[6] binding. The molecular relay was reprogrammed to detect avidin by using a different TFG. Finally, Xe binding by CB[6] was detected in buffer and in E. coli cultures expressing CAII through ultrasensitive (129) Xe NMR spectroscopy.


Assuntos
Espectroscopia de Ressonância Magnética/métodos , Xenônio/química , Cristalografia por Raios X , Limite de Detecção
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