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1.
Opt Express ; 31(18): 29107-29118, 2023 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-37710717

RESUMO

The attenuation coefficient of natural waters plays a significant role in our understanding of hydrology from both the oceanographic and biological point of view. The advent of near-continuous observations by sophisticated space-based lidars now offers an unprecedented opportunity to characterize attenuation coefficients over open oceans on global and regional scales. At present, however, literature reports of lidar-derived attenuation coefficient estimates (klidar, m-1) in oceanic waters are very limited. In this study, we present a global survey of klidar derived from ATLAS/ICESat-2 nighttime measurements. Our results augment the existing passive sensor ocean color data set with a new diurnal component and extend the record to now include previously unavailable polar nighttime observations. The values of ATLAS measured klidar at 532 nm are between 0.045 and 0.39 m-1 with the higher values (>0.15 m-1) correlated with coastal waters and sea ice covered oceans. The average klidar in clearest oligotrophic ocean gyres is ∼0.058 ± 0.012 m-1 at 532 nm. The results reported here demonstrate the feasibility of using ATLAS/ICESat-2 lidar measurements for global klidar studies, which will in turn provide critical insights that enable climate models to correctly describe the amount of light present under sea ice, and for heat deposition studies in the upper ocean.

2.
Opt Express ; 30(20): 36509-36525, 2022 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-36258578

RESUMO

In shallow nearshore waters, seafloor heights and properties can be accurately measured by the current generation of space-based elastic backscatter lidars: CALIOP, flying aboard the CALIPSO satellite and ATLAS aboard ICESat-2. CALIOP's 532 nm volume depolarization ratios, together with the ratios of the attenuated backscatter coefficients measured at 532 nm and 1064 nm, can efficiently distinguish optically shallow waters from nearby land surfaces and deep oceans. ATLAS's high vertical resolution photon measurements can accurately determine seafloor depths in shallow water bodies, characterize seafloor reflectance, and provide assessments of ocean biomass concentrations in the intervening water column. By adding bathymetry, seafloor optical properties (e.g., reflectance, depolarization ratio and attenuated backscatter), and nighttime observations, space lidar measurements obtained in nearshore waters can provide a wealth of unique information to complement existing satellite-based ocean color remote sensing capabilities. The results reported here demonstrate the feasibility of using satellite lidars for nearshore seafloor ecosystem analyses, which in turn provide critical insights for studies of coastal navigation and seabed topography changes due to disasters, as well as the temporal and spatial morphological evolution of coastal systems.

3.
Opt Express ; 24(1): 620-36, 2016 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-26832292

RESUMO

An invariant imbedding T-matrix (II-TM) method is used to calculate the single-scattering properties of 8-column aggregate ice crystals. The II-TM based backscatter values are compared with those calculated by the improved geometric-optics method (IGOM) to refine the backscattering properties of the ice cloud radiative model used in the MODIS Collection 6 cloud optical property product. The integrated attenuated backscatter-to-cloud optical depth (IAB-ICOD) relation is derived from simulations using a CALIPSO (Cloud-Aerosol Lidar and Infrared Pathfinder Satellite) lidar simulator based on a Monte Carlo radiative transfer model. By comparing the simulation results and co-located CALIPSO and MODIS (Moderate Resolution Imaging Spectroradiometer) observations, the non-uniform zonal distribution of ice clouds over ocean is characterized in terms of a mixture of smooth and rough ice particles. The percentage of the smooth particles is approximately 6% and 9% for tropical and midlatitude ice clouds, respectively.

4.
Org Biomol Chem ; 14(38): 8942-8946, 2016 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-27722405

RESUMO

Bioincorporation of the methionine analogue S-(2-fluoroethyl)-l-homocysteine (l-MFE) into bacteriophage lysozyme overproduced in Escherichia coli results not only in the expected l-MFE incorporation but surprisingly substantial l-vinthionine incorporation into the labeled lysozymes. Synthetic l-vinthionine itself however is not activated by purified Escherichia coli methionyl-tRNA synthetase. The indirect preparation of vinthionine-containing proteins has the potential to be an alternate strategy to prepare vinyl thioether moieties for click chemistry applications on proteins.


Assuntos
Aminoácidos/metabolismo , Bacteriófago lambda/metabolismo , Escherichia coli/metabolismo , Escherichia coli/virologia , Metionina/análogos & derivados , Muramidase/metabolismo , Proteínas Virais/metabolismo , Aminoácidos/análise , Bacteriófago lambda/química , Escherichia coli/química , Proteínas de Escherichia coli/análise , Proteínas de Escherichia coli/metabolismo , Etionina/análogos & derivados , Etionina/análise , Etionina/metabolismo , Halogenação , Homocisteína/análogos & derivados , Homocisteína/análise , Homocisteína/metabolismo , Metionina/análise , Metionina/metabolismo , Metionina tRNA Ligase/análise , Metionina tRNA Ligase/metabolismo , Modelos Moleculares , Muramidase/análise , Biossíntese de Proteínas , Proteínas Virais/análise
5.
Earth Space Sci ; 8(6): e2021EA001729, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34222563

RESUMO

The Advanced Topographic Laser Altimeter System (ATLAS) aboard the Ice, Cloud, and land Elevation Satellite-2 (ICESat-2) has been making very high resolution measurements of the Earth's surface elevation since October 2018. ATLAS uses photomultiplier tubes (PMTs) as detectors in photon counting mode, so that a single photon reflected back to the receiver triggers a detection within the ICESat-2 data acquisition system. However, one characteristic of ICESat-2 detected photons is the possible presence of afterpulses, defined as small amplitude pulses occurring after the primary signal pulse due to photon arrival. The disadvantage of these afterpulses is that they often confound the accurate measurements of low level signals following a large amplitude of signal and can degrade energy resolution and cause errors in pulse counting applications. This paper discusses and summarizes the after-pulsing effects exhibited by the ATLAS PMTs based on on-orbit measurements over different seasons and geographic regions. The potential impacts of these after-pulsing effects on altimetry and ocean subsurface retrievals are discussed.

6.
Atmos Meas Tech ; 12(11): 6241-6258, 2019 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-33414857

RESUMO

The Cloud-Aerosol Transport System (CATS) lidar on board the International Space Station (ISS) operated from 10 February 2015 to 30 October 2017 providing range-resolved vertical backscatter profiles of Earth's atmosphere at 1064 and 532 nm. The CATS instrument design and ISS orbit lead to a higher 1064 nm signal-to-noise ratio than previous space-based lidars, allowing for direct atmospheric calibration of the 1064 nm signals. Nighttime CATS Version 3-00 data were calibrated by scaling the measured data to a model of the expected atmospheric backscatter between 22 and 26 km above mean sea level (AMSL). The CATS atmospheric model is constructed using molecular backscatter profiles derived from Modern-Era Retrospective analysis for Research and Applications, Version 2 (MERRA-2) re-analysis data and aerosol scattering ratios measured by the Cloud-Aerosol Lidar with Orthogonal Polarization (CALIOP). The nighttime normalization altitude region was chosen to simultaneously minimize aerosol loading and variability within the CATS data frame, which extends from 28 km to -2 km AMSL. Daytime CATS Version 3-00 data were calibrated through comparisons with nighttime measurements of the layer integrated attenuated total backscatter (iATB) from strongly scattering, rapidly attenuating opaque cirrus clouds. The CATS nighttime 1064 nm attenuated total backscatter (ATB) uncertainties for clouds and aerosols are primarily related to the uncertainties in the CATS nighttime calibration technique, which are estimated to be ~9%. Median CATS V3-00 1064 nm ATB relative uncertainty at night within cloud and aerosol layers is 7%, slightly lower than these calibration uncertainty estimates. CATS median daytime 1064 nm ATB relative uncertainty is 21% in cloud and aerosol layers, similar to the estimated 16-18% uncertainty in the CATS daytime cirrus cloud calibration transfer technique. Coincident daytime comparisons between CATS and the Cloud Physics Lidar (CPL) during the CATS-CALIPSO Airborne Validation Experiment (CCAVE) project show good agreement in mean ATB profiles for clear-air regions. Eight nighttime comparisons between CATS and the PollyXT ground based lidars also show good agreement in clear-air regions between 3-12 km, with CATS having a mean ATB of 19.7 % lower than PollyXT. Agreement between the two instruments (~7%) is even better within an aerosol layer. Six-month comparisons of nighttime ATB values between CATS and the Cloud-Aerosol Lidar with Orthogonal Polarization (CALIOP) also show that iATB comparisons of opaque cirrus clouds agree to within 19%. Overall, CATS has demonstrated that direct calibration of the 1064 nm channel is possible from a space based lidar using the atmospheric normalization technique.

7.
Atmos Meas Tech ; 11(7): 4129-4152, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33510819

RESUMO

The CALIPSO (Cloud-Aerosol Lidar and Infrared Pathfinder Satellite Observations) level 3 aerosol profile product reports globally gridded, quality-screened, monthly mean aerosol extinction profiles retrieved by CALIOP (the Cloud-Aerosol Lidar with Orthogonal Polarization). This paper describes the quality screening and averaging methods used to generate the version 3 product. The fundamental input data are CALIOP level 2 aerosol extinction profiles and layer classification information (aerosol, cloud, and clear-air). Prior to aggregation, the extinction profiles are quality-screened by a series of filters to reduce the impact of layer detection errors, layer classification errors, extinction retrieval errors, and biases due to an intermittent signal anomaly at the surface. The relative influence of these filters are compared in terms of sample rejection frequency, mean extinction, and mean aerosol optical depth (AOD). The "extinction QC flag" filter is the most influential in preventing high-biases in level 3 mean extinction, while the "misclassified cirrus fringe" filter is most aggressive at rejecting cirrus misclassified as aerosol. The impact of quality screening on monthly mean aerosol extinction is investigated globally and regionally. After applying quality filters, the level 3 algorithm calculates monthly mean AOD by vertically integrating the monthly mean quality-screened aerosol extinction profile. Calculating monthly mean AOD by integrating the monthly mean extinction profile prevents a low bias that would result from alternately integrating the set of extinction profiles first and then averaging the resultant AOD values together. Ultimately, the quality filters reduce level 3 mean AOD by -24 and -31% for global ocean and global land, respectively, indicating the importance of quality screening.

8.
Atmos Meas Tech ; 11(6): 3281-3296, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33868503

RESUMO

This paper presents an innovative retrieval method that translate the CALIOP land surface laser pulse returns into the surface bidirectional reflectance. To better analyze the surface returns, the CALIOP receiver impulse response and the downlinked samples' distribution at 30 m resolution are discussed. The saturated laser pulse returns from snow and ice surfaces are recovered based on surface tail information. The retrieved snow surface bidirectional reflectance is compared with reflectance from both CALIOP cloud cover regions and MODIS BRDF/Albedo model parameters. Besides the surface bidirectional reflectance, the column top-of-atmosphere bidirectional reflectance is calculated from the CALIOP lidar background data. It is compared with bidirectional reflectance from WFC radiance measurements. The retrieved CALIOP surface bidirectional reflectance and column top-of-atmosphere bidirectional reflectance results provide unique information to complement existing MODIS standard data products and would have valuable applications for modellers.

9.
Atmos Meas Tech ; 11(1): 499-514, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33868502

RESUMO

Due to instrument sensitivities and algorithm detection limits, level 2 (L2) Cloud-Aerosol Lidar with Orthogonal Polarization (CALIOP) 532nm aerosol extinction profile retrievals are often populated with retrieval fill values (RFVs), which indicate the absence of detectable levels of aerosol within the profile. In this study, using 4 years (2007-2008 and 2010-2011) of CALIOP version 3 L2 aerosol data, the occurrence frequency of daytime CALIOP profiles containing all RFVs (all-RFV profiles) is studied. In the CALIOP data products, the aerosol optical thickness (AOT) of any all-RFV profile is reported as being zero, which may introduce a bias in CALIOP-based AOT climatologies. For this study, we derive revised estimates of AOT for all-RFV profiles using collocated Moderate Resolution Imaging Spectroradiometer (MODIS) Dark Target (DT) and, where available, AErosol RObotic NEtwork (AERONET) data. Globally, all-RFV profiles comprise roughly 71% of all daytime CALIOP L2 aerosol profiles (i.e., including completely attenuated profiles), accounting for nearly half (45 %) of all daytime cloud-free L2 aerosol profiles. The mean collocated MODIS DT (AERONET) 550 nm AOT is found to be near 0.06 (0.08) for CALIOP all-RFV profiles. We further estimate a global mean aerosol extinction profile, a so-called "noise floor", for CALIOP all-RFV profiles. The global mean CALIOP AOT is then recomputed by replacing RFV values with the derived noise-floor values for both all-RFV and non-all-RFV profiles. This process yields an improvement in the agreement of CALIOP and MODIS over-ocean AOT.

10.
Atmos Meas Tech ; 11(11): 6107-6135, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-31921372

RESUMO

The Cloud-Aerosol Lidar with Orthogonal Polarization (CALIOP) version 4.10 (V4) level 2 aerosol data products, released in November 2016, include substantial improvements to the aerosol subtyping and lidar ratio selection algorithms. These improvements are described along with resulting changes in aerosol optical depth (AOD). The most fundamental change in V4 level 2 aerosol products is a new algorithm to identify aerosol subtypes in the stratosphere. Four aerosol subtypes are introduced for the stratospheric aerosols: polar stratospheric aerosol (PSA), volcanic ash, sulfate/other, and smoke. The tropospheric aerosol subtyping algorithm was also improved by adding the following enhancements: (1) all aerosol subtypes are now allowed over polar regions, whereas the version 3 (V3) algorithm allowed only clean continental and polluted continental aerosols; (2) a new "dusty marine" aerosol subtype is introduced, representing mixtures of dust and marine aerosols near the ocean surface; and (3) the "polluted continental" and "smoke" subtypes have been renamed "polluted continental/smoke" and "elevated smoke", respectively. V4 also revises the lidar ratios for clean marine, dust, clean continental, and elevated smoke subtypes. As a consequence of the V4 updates, the mean 532 nm AOD retrieved by CALIOP has increased by 0.044 (0.036) or 52 % (40 %) for nighttime (daytime). Lidar ratio revisions are the most influential factor for AOD changes from V3 to V4, especially for cloud-free skies. Preliminary validation studies show that the AOD discrepancies between CALIOP and AERONET/MODIS (ocean) are reduced in V4 compared to V3.

11.
Atmos Meas Tech ; 11(3): 1459-1479, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33479568

RESUMO

Data products from the Cloud-Aerosol Lidar with Orthogonal Polarization (CALIOP) on board Cloud-Aerosol Lidar and Infrared Pathfinder Satellite Observations (CALIPSO) were recently updated following the implementation of new (version 4) calibration algorithms for all of the level 1 attenuated backscatter measurements. In this work we present the motivation for and the implementation of the version 4 nighttime 532 nm parallel channel calibration. The nighttime 532 nm calibration is the most fundamental calibration of CALIOP data, since all of CALIOP's other radiometric calibration procedures - i.e., the 532 nm daytime calibration and the 1064 nm calibrations during both nighttime and daytime - depend either directly or indirectly on the 532 nm nighttime calibration. The accuracy of the 532 nm nighttime calibration has been significantly improved by raising the molecular normalization altitude from 30-34 km to 36-39 km to substantially reduce stratospheric aerosol contamination. Due to the greatly reduced molecular number density and consequently reduced signal-to-noise ratio (SNR) at these higher altitudes, the signal is now averaged over a larger number of samples using data from multiple adjacent granules. As well, an enhanced strategy for filtering the radiation-induced noise from high energy particles was adopted. Further, the meteorological model used in the earlier versions has been replaced by the improved MERRA-2 model. An aerosol scattering ratio of 1.01 ± 0.01 is now explicitly used for the calibration altitude. These modifications lead to globally revised calibration coefficients which are, on average, 2-3% lower than in previous data releases. Further, the new calibration procedure is shown to eliminate biases at high altitudes that were present in earlier versions and consequently leads to an improved representation of stratospheric aerosols. Validation results using airborne lidar measurements are also presented. Biases relative to collocated measurements acquired by the Langley Research Center (LaRC) airborne high spectral resolution lidar (HSRL) are reduced from 3.6% ± 2.2% in the version 3 data set to 1.6% ± 2.4 % in the version 4 release.

12.
Curr Opin Chem Biol ; 10(5): 509-19, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16905354

RESUMO

In recent years, substantial advances have been made in the engineering of glycosidases and glycosyltransferases for the synthesis and degradation of glycan structures. Key developments include improvement of the thermostability of xylanase through comprehensive saturation mutagenesis, creation of the first glycosynthase derived from an inverting glycosidase and the emergence of a new class of modified glycosidases capable of efficiently synthesizing thioglycosidic linkages. Of particular note is the increased use of random mutagenesis and directed evolution tactics for tailoring glycosidase activity. Although the engineering of glycosyltransferases is still in its early stages, recent work on the structure-based alteration of substrate specificity and the manipulation of glycosyltransferase profiles in whole cells to effect complex changes in in vivo glycobiology probably foreshadows a wave of considerable innovation in this area.


Assuntos
Glicosídeo Hidrolases/química , Glicosiltransferases/química , Engenharia de Proteínas , Animais , Catálise , Ativação Enzimática , Humanos , Polissacarídeos/síntese química , Polissacarídeos/química
13.
Opt Express ; 15(9): 5327-32, 2007 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-19532786

RESUMO

Using measurements obtained by the Cloud-Aerosol Lidar and Infrared Pathfinder Satellite Observations (CALIPSO) satellite, relationships between layer-integrated depolarization ratio (delta) and layer-integrated attenuated backscatter (gamma) are established for moderately thick clouds of both ice and water. A new and simple form of the delta-gamma relation for spherical particles, developed from Monte Carlo simulations and suitable for both water clouds and spherical aerosol particles, is found to agree well with the observations. A high-backscatter, low-depolarization delta-gamma relationship observed for some ice clouds is shown to result primarily from horizontally oriented plates and implies a preferential lidar ratio - depolarization ratio relation in nature for ice cloud particles containing plates.

14.
Opt Express ; 15(22): 14504-15, 2007 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-19550729

RESUMO

A technique we refer to as Elevation Information in Tail (EIT) has been developed to provide improved lidar altimetry from CALIPSO lidar data. The EIT technique is demonstrated using CALIPSO data and is applicable to other similar lidar systems with low-pass filters. The technique relies on an observed relation between the shape of the surface return signals (peak shape) and the detector photo-multiplier tube transient response (transient response tail). Application of the EIT to CALIPSO data resulted in an order of magnitude or better improvement in the CALIPSO land surface 30-meter elevation measurements. The results of EIT compared very well with the National Elevation Database (NED) high resolution elevation maps, and with the elevation measurements from the Shuttle Radar Topography Mission (SRTM).

15.
J Geophys Res Atmos ; 122(2): 1098-1113, 2017 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-31534879

RESUMO

The CALIOP data processing scheme only retrieves extinction profiles in those portions of the return signal where cloud or aerosol layers have been identified by the CALIOP layer detection scheme. In this study we use two years of CALIOP and MODIS data to quantify the aerosol optical depth of undetected weakly backscattering layers. Aerosol extinction and column-averaged lidar ratio is retrieved from CALIOP Level 1B (Version 4) profile using MODIS AOD as a constraint over oceans from March 2013 to February 2015. To quantify the undetected layer AOD (ULA), an unconstrained retrieval is applied globally using a lidar ratio of 28.75 sr estimated from constrained retrievals during the daytime over the ocean. We find a global mean ULA of 0.031 ± 0.052. There is no significant difference in ULA between land and ocean. However, the fraction of undetected aerosol layers rises considerably during daytime, when the large amount of solar background noise lowers the signal to noise ratio (SNR). For this reason, there is a difference in ULA between day (0.036 ± 0.066) and night (0.025 ± 0.021). ULA is larger in the northern hemisphere and relatively larger at high latitudes. Large ULA for the Polar Regions is strongly related to the cases where the CALIOP Level 2 Product reports zero AOD. This study provides an estimate of the complement of AOD that is not detected by lidar, and bounds the CALIOP AOD uncertainty to provide corrections for science studies that employ the CALIOP Level 2 AOD.

16.
J Mol Biol ; 332(1): 59-72, 2003 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-12946347

RESUMO

Binding of methionine to methionyl-tRNA synthetase (MetRS) is known to promote conformational changes within the active site. However, the contribution of these rearrangements to enzyme catalysis is not fully understood. In this study, several methionine and methionyl adenylate analogues were diffused into crystals of the monomeric form of Escherichia coli methionyl-tRNA synthetase. The structures of the corresponding complexes were solved at resolutions below 1.9A and compared to those of the enzyme free or complexed with methionine. Residues Y15 and W253 play key roles in the strength of the binding of the amino acid and of its analogues. Indeed, full motions of these residues are required to recover the maximum in free energy of binding. Residue Y15 also controls the size of the hydrophobic pocket where the amino acid side-chain interacts. H301 appears to participate to the specific recognition of the sulphur atom of methionine. Complexes with methionyl adenylate analogues illustrate the shielding by MetRS of the region joining the methionine and adenosine moieties. Finally, the structure of MetRS complexed to a methionine analogue mimicking the tetrahedral carbon of the transition state in the aminoacylation reaction was solved. On the basis of this model, we propose that, in response to the binding of the 3'-end of tRNA, Y15 moves again in order to deshield the anhydride bond in the natural adenylate.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/química , Escherichia coli/metabolismo , Metionina tRNA Ligase/química , Metionina/análogos & derivados , Metionina/química , Conformação Proteica , Monofosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Catálise , Cristalografia por Raios X , Metionina/metabolismo , Metionina tRNA Ligase/metabolismo , Dados de Sequência Molecular , Estrutura Molecular , Ligação Proteica , Alinhamento de Sequência
17.
Curr Med Chem ; 9(3): 385-409, 2002 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11860363

RESUMO

The increasing need for new antibiotics to overcome rapidly developing resistance mechanisms observed in clinical isolates of Gram-positive and Gram-negative eubacteria has placed critical emphasis on the search for new antibacterial enzyme targets and the structural and mechanistic investigation of such targets. Among these potential targets, the enzymes responsible for integrating the amino acid methionine into proteins, along with its subsequent post-translational modification and repair, have emerged as promising candidates for the development of novel antibiotics. As well, there is increasing evidence for the importance of several of these enzymes in the development of anti-cancer, anti-parasitic, and anti-atherosclerotic drugs. Within the last three years, the crystal structures of all of these enzymes have been determined, which offers an unprecedented source of structural information for inhibitor design. The development of combinatorial chemistry and high throughput screening procedures has quickly provided several potent, specific inhibitors for a number of these enzymes, particularly the peptide deformylase, methionine aminopeptidase, and methionyl-tRNA synthetase enzymes. This review critically analyzes the future potential for inhibition of enzymes in this pathway, allowing for a pragmatic view of the success of inhibitor developments and highlighting areas in which further investigations are warranted.


Assuntos
Amidoidrolases , Desenho de Fármacos , Inibidores Enzimáticos/química , Metionina/metabolismo , Aminopeptidases/antagonistas & inibidores , Animais , Bovinos , Escherichia coli/enzimologia , Humanos , Hidroximetil e Formil Transferases/antagonistas & inibidores , Metionina Sulfóxido Redutases , Metionina tRNA Ligase/antagonistas & inibidores , Metionil Aminopeptidases , Modelos Moleculares , Oxirredutases/antagonistas & inibidores , Ligação Proteica , Relação Estrutura-Atividade
18.
J Biomol Struct Dyn ; 21(2): 235-46, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12956607

RESUMO

The leucine-isoleucine-valine binding protein (LIV) found in the periplasmic space of E. coli has been used as a structural model for a number of neuronal receptors. This "venus fly trap" type protein has been characterized by crystallography in only the open form. Herein we have labeled LIV with 5-fluorotryptophan (5F-Trp) and difluoromethionine (DFM) in order to explore the structural dynamics of this protein and the application of DFM as a potential (19)F NMR structural probe for this family of proteins. Based on mass spectrometric analysis of the protein overproduced in the presence of DFM, approximately 30% of the five LIV methionine residues were randomly substituted with the fluorinated analog. Urea denaturation experiments imply a slight decrease in protein stability when DFM is incorporated into LIV. However, the fluorinated methionine did not alter leucine-binding activity upon its incorporation into the protein. Binding of L-leucine stabilizes both the unlabeled and DFM-labeled LIV, and induces the protein to adopt a three-state unfolding model in place of the two-state process observed for the free protein. The (19)F NMR spectrum of DFM-labeled LIV gave distinct resonances for the five Met residues found in LIV. 5F-Trp labeled LIV gave a well resolved spectrum for the three Trp residues. Trp to Phe mutants defined the resonances in the spectrum. The distinct narrowing in line width of the resonances when ligand was added identified the closed form of the protein.


Assuntos
Proteínas de Bactérias , Proteínas de Transporte/química , Flúor/química , Conformação Proteica , Soluções , Proteínas de Escherichia coli/química , Matemática , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Desnaturação Proteica , Triptofano/química
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