RESUMEN
The versatile functions of fluorescent proteins (FPs) as fluorescence biomarkers depend on their intrinsic chromophores interacting with the protein environment. Besides X-ray crystallography, vibrational spectroscopy represents a highly valuable tool for characterizing the chromophore structure and revealing the roles of chromophore-environment interactions. In this work, we aim to benchmark the ground-state vibrational signatures of a series of FPs with emission colors spanning from green, yellow, orange, to red, as well as the solvated model chromophores for some of these FPs, using wavelength-tunable femtosecond stimulated Raman spectroscopy (FSRS) in conjunction with quantum calculations. We systematically analyzed and discussed four factors underlying the vibrational properties of FP chromophores: sidechain structure, conjugation structure, chromophore conformation, and the protein environment. A prominent bond-stretching mode characteristic of the quinoidal resonance structure is found to be conserved in most FPs and model chromophores investigated, which can be used as a vibrational marker to interpret chromophore-environment interactions and structural effects on the electronic properties of the chromophore. The fundamental insights gained for these light-sensing units (e.g., protein active sites) substantiate the unique and powerful capability of wavelength-tunable FSRS in delineating FP chromophore properties with high sensitivity and resolution in solution and protein matrices. The comprehensive characterization for various FPs across a colorful palette could also serve as a solid foundation for future spectroscopic studies and the rational engineering of FPs with diverse and improved functions.
Asunto(s)
Espectrometría Raman , Espectrometría Raman/métodos , Proteínas Fluorescentes Verdes/metabolismo , Dominio CatalíticoRESUMEN
Fluorescent noncanonical amino acids (fNCAAs) could serve as starting points for the rational design of protein-based fluorescent sensors of biological activity. However, efforts toward this goal are likely hampered by a lack of atomic-level characterization of fNCAAs within proteins. Here, we describe the spectroscopic and structural characterization of five streptavidin mutants that contain the fNCAA l-(7-hydroxycoumarin-4-yl)ethylglycine (7-HCAA) at sites proximal to the binding site of its substrate, biotin. Many of the mutants exhibited altered fluorescence spectra in response to biotin binding, which included both increases and decreases in fluorescence intensity as well as red- or blue-shifted emission maxima. Structural data were also obtained for three of the five mutants. The crystal structures shed light on interactions between 7-HCAA and functional groups, contributed either by the protein or by the substrate, that may be responsible for the observed changes in the 7-HCAA spectra. These data could be used in future studies aimed at the rational design of fluorescent, protein-based sensors of small molecule binding or dissociation.
Asunto(s)
Aminoácidos/química , Biotina/química , Proteínas Recombinantes/química , Estreptavidina/química , Sitios de Unión , Fenómenos Biofísicos , Cristalografía por Rayos X/métodos , Fluorescencia , Ligandos , Modelos Moleculares , Conformación Proteica , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Genetically encoded fluorescent noncanonical amino acids (fNCAAs) could be used to develop novel fluorescent sensors of protein function. Previous efforts toward this goal have been limited by the lack of extensive physicochemical and structural characterizations of protein-based sensors containing fNCAAs. Here, we report the steady-state spectroscopic properties and first structural analyses of an fNCAA-containing Fab fragment of the 5c8 antibody, which binds human CD40L. A previously reported 5c8 variant in which the light chain residue IleL98 is replaced with the fNCAA l-(7-hydroxycoumarin-4-yl)ethylglycine (7-HCAA) exhibits a 1.7-fold increase in fluorescence upon antigen binding. Determination and comparison of the apparent pKas of 7-HCAA in the unbound and bound forms indicate that the observed increase in fluorescence is not the result of perturbations in pKa. Crystal structures of the fNCAA-containing Fab in the apo and bound forms reveal interactions between the 7-HCAA side chain and surrounding residues that are disrupted upon antigen binding. This structural characterization not only provides insight into the manner in which protein environments can modulate the fluorescence properties of 7-HCAA but also could serve as a starting point for the rational design of new fluorescent protein-based reporters of protein function.
Asunto(s)
Aminoácidos/química , Sitios de Unión de Anticuerpos , Ligando de CD40/química , Fluorescencia , Colorantes Fluorescentes/química , Fragmentos Fab de Inmunoglobulinas/química , Aminoácidos/metabolismo , Ligando de CD40/metabolismo , Cristalografía por Rayos X , Humanos , Fragmentos Fab de Inmunoglobulinas/metabolismo , Modelos Moleculares , Conformación ProteicaRESUMEN
Higher-plant Rubisco activase (Rca) plays a critical role in regulating the activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). In vitro, Rca is known to undergo dynamic assembly-disassembly processes, with several oligomer stoichiometries coexisting over a broad concentration range. Although the hexamer appears to be the active form, changes in quaternary structure could play a role in Rubisco regulation. Therefore, fluorescent labels were attached to the C-termini of spinach ß-Rca, and the rate of subunit mixing was monitored by measuring energy transfer as a function of nucleotide and divalent cation. Only dimeric units appeared to exchange. Poorly hydrolyzable substrate analogues provided locked complexes with high thermal stabilities (apparent Tm = 60 °C) and an estimated t1/2 of at least 7 h, whereas ATP-Mg provided tight assemblies with t1/2 values of 30-40 min and ADP-Mg loose assemblies with t1/2 values of <15 min. Accumulation of ADP to 20% of the total level of adenine nucleotide substantially accelerated equilibration. An initial lag period was observed with ATP·Mg, indicating inhibition of subunit exchange at low ADP concentrations. The ADP Ki value was estimated to exceed the Km for ATP (0.772 ± 96 mM), suggesting that the equilibration rate is a function of the relative contributions of high- and low-affinity states. C-Terminal cross-linking generated covalent dimers, required the N-terminal extension to the AAA+ domain, and provided evidence of different classes of sites. We propose that oligomer reorganization may be stalled during periods of high Rubisco reactivation activity, whereas changes in quaternary structure are stimulated by the accumulation of ADP at low light levels.
Asunto(s)
Proteínas de Plantas/metabolismo , Spinacia oleracea/enzimología , Adenosina Difosfato , Adenosina Trifosfato , Clonación Molecular , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Modelos Moleculares , Proteínas de Plantas/genética , Conformación Proteica , Multimerización de Proteína , Subunidades de ProteínaRESUMEN
In many photosynthetic organisms, tight-binding Rubisco inhibitors are released by the motor protein Rubisco activase (Rca). In higher plants, Rca plays a pivotal role in regulating CO2 fixation. Here, the ATPase activity of 0.005 mm tobacco Rca was monitored under steady-state conditions, and global curve fitting was utilized to extract kinetic constants. The kcat was best fit by 22.3 ± 4.9 min(-1), the Km for ATP by 0.104 ± 0.024 mm, and the Ki for ADP by 0.037 ± 0.007 mm. Without ADP, the Hill coefficient for ATP hydrolysis was extracted to be 1.0 ± 0.1, indicating noncooperative behavior of homo-oligomeric Rca assemblies. However, the addition of ADP was shown to introduce positive cooperativity between two or more subunits (Hill coefficient 1.9 ± 0.2), allowing for regulation via the prevailing ATP/ADP ratio. ADP-mediated activation was not observed, although larger amounts led to competitive product inhibition of hydrolytic activity. The catalytic efficiency increased 8.4-fold upon cooperative binding of a second magnesium ion (Hill coefficient 2.5 ± 0.5), suggesting at least three conformational states (ATP-bound, ADP-bound, and empty) within assemblies containing an average of about six subunits. The addition of excess Rubisco (24:1, L8S8/Rca6) and crowding agents did not modify catalytic rates. However, high magnesium provided for thermal Rca stabilization. We propose that magnesium mediates the formation of closed hexameric toroids capable of high turnover rates and amenable to allosteric regulation. We suggest that in vivo, the Rca hydrolytic activity is tuned by fluctuating [Mg(2+)] in response to changes in available light.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Gossypium/enzimología , Magnesio/química , Nicotiana/enzimología , Ribulosa-Bifosfato Carboxilasa/metabolismo , Adenosina Difosfato/química , Adenosina Trifosfatasas/química , Adenosina Trifosfato/química , Sitio Alostérico , Catálisis , Dominio Catalítico , Cloroplastos/enzimología , Hidrólisis , Luz , NAD/química , Oxidación-Reducción , Fotosíntesis , Unión ProteicaRESUMEN
We report a fluorescence correlation spectroscopy (FCS) study of the assembly pathway of the AAA+ protein ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase (Rca), a ring-forming ATPase responsible for activation of inhibited Rubisco complexes for biological carbon fixation. A thermodynamic characterization of simultaneously populated oligomeric states appears critical in understanding Rca structure and function. Using cotton ß-Rca, we demonstrate that apparent diffusion coefficients vary as a function of concentration, nucleotide, and cation. Using manual fitting procedures, we provide estimates for the equilibrium constants for the stepwise assembly and find that in the presence of ATPγS, the Kd for hexamerization is 10-fold lower than with ADP (â¼0.1 vs â¼1 µM). Hexamer fractions peak at 30 µM and dominate at 8-70 µM Rca, where they comprise 60-80% of subunits with ATPγS, compared with just 30-40% with ADP. Dimer fractions peak at 1-4 µM Rca, where they comprise 15-18% with ATPγS and 26-28% with ADP. At 30 µM Rca, large aggregates begin to form that comprise â¼10% of total protein with ATPγS and â¼25% with ADP. FCS data collected on the catalytically impaired WalkerB-D173N variant in the presence of ATP provided strong support for these results. Titration with free magnesium ions lead to the disaggregation of larger complexes in favor of hexameric forms, suggesting that a second magnesium binding site with a Kd value of 1-3 mM mediates critical subunit contacts. We propose that closed-ring toroidal hexameric forms are stabilized by binding of Mg·ATP plus Mg2+, whereas Mg·ADP promotes continuous assembly to supramolecular aggregates such as spirals.
Asunto(s)
Adenosina Trifosfato/análogos & derivados , Gossypium/enzimología , Magnesio/metabolismo , Ribulosa-Bifosfato Carboxilasa/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Gossypium/química , Gossypium/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Multimerización de Proteína , Estabilidad Proteica , Ribulosa-Bifosfato Carboxilasa/químicaRESUMEN
Rubisco activase (Rca) is a chaperone-like protein of the AAA+ family, which uses mechano-chemical energy derived from ATP hydrolysis to release tightly bound inhibitors from the active site of the primary carbon fixing enzyme ribulose 1,5-bisphosphate oxygenase/carboxylase (Rubisco). Mechanistic and structural investigations of Rca have been hampered by its exceptional thermolability, high degree of size polydispersity and propensity towards subunit aggregation. In this work, we have characterized the thermal stability and self-association behavior of recombinant Rca preparations, and have developed ligand screening methods. Thermal denaturation profiles generated by circular dichroism indicate that creosote and tobacco short-form Rcas are the most stable proteins examined, with an estimated mid-point temperature of 45-47°C for protein denaturation. We demonstrate that ADP provides a higher degree of stabilization than ATP, that magnesium ions have a small stabilizing effect on ATP-bound, but a significant destabilizing effect on ADP-bound Rca, and that phosphate provides weak stabilization of the ADP-bound form of the protein. A dimeric species was identified by size-exclusion chromatography, suggesting that the two-subunit module may comprise the basic building block for larger assemblies. Evidence is provided that chromatographic procedures reflect non-equilibrium multimeric states. Dynamic light scattering experiments performed on nucleotide-bearing Rca support the notion that several larger, highly polydisperse assembly states coexist over a broad concentration range. No significant changes in aggregation are observed upon replacement of ADP with ATP. However, in the absence of nucleotides, the major protein population appears to consist of a monodisperse oligomer smaller than a hexamer.
Asunto(s)
Larrea/química , Chaperonas Moleculares/química , Nicotiana/química , Proteínas de Plantas/química , Multimerización de Proteína , Adenosina Trifosfato/química , Dicroismo Circular , Desnaturalización Proteica , Estabilidad Proteica , Estructura Cuaternaria de ProteínaRESUMEN
Photochromic fluorescent proteins (FPs) have proved to be indispensable luminous probes for sophisticated and advanced bioimaging techniques. Among them, an interplay between photoswitching and photoconversion has only been observed in a limited subset of Kaede-like FPs that show potential for discovering the key mechanistic steps during green-to-red photoconversion. Various spectroscopic techniques including femtosecond stimulated Raman spectroscopy (FSRS), X-ray crystallography, and femtosecond transient absorption were employed on a set of five related FPs with varying photoconversion and photoswitching efficiencies. A 3-methyl-histidine chromophore derivative, incorporated through amber suppression using orthogonal aminoacyl tRNA synthetase/tRNA pairs, displays more dynamic photoswitching but greatly reduced photoconversion versus the least-evolved ancestor (LEA). Excitation-dependent measurements of the green anionic chromophore reveal that the varying photoswitching efficiencies arise from both the initial transient dynamics of the bright cis state and the final trans-like photoswitched off state, with an exocyclic bridge H-rocking motion playing an active role during the excited-state energy dissipation. This investigation establishes a close-knit feedback loop between spectroscopic characterization and protein engineering, which may be especially beneficial to develop more versatile FPs with targeted mutations and enhanced functionalities, such as photoconvertible FPs that also feature photoswitching properties.
RESUMEN
The rapid release of tight-binding inhibitors from dead-end ribulose-bisphosphate carboxylase/oxygenase (Rubisco) complexes requires the activity of Rubisco activase, an AAA+ ATPase that utilizes chemo-mechanical energy to catalyze the reactivation of Rubisco. Activase is thought to play a central role in coordinating the rate of CO(2) fixation with the light reactions of photosynthesis. Here, we present a 1.9 Å crystal structure of the C-domain core of creosote activase. The fold consists of a canonical four-helix bundle, from which a paddle-like extension protrudes that entails a nine-turn helix lined by an irregularly structured peptide strand. The residues Lys-313 and Val-316 involved in the species-specific recognition of Rubisco are located near the tip of the paddle. An ionic bond between Lys-313 and Glu-309 appears to stabilize the glycine-rich end of the helix. Structural superpositions onto the distant homolog FtsH imply that the paddles extend away from the hexameric toroid in a fan-like fashion, such that the hydrophobic sides of each blade bearing Trp-302 are facing inward and the polar sides bearing Lys-313 and Val-316 are facing outward. Therefore, we speculate that upon binding, the activase paddles embrace the Rubisco cylinder by placing their hydrophobic patches near the partner protein. This model suggests that conformational adjustments at the remote end of the paddle may relate to selectivity in recognition, rather than specific ionic contacts involving Lys-313. Additionally, the superpositions predict that the catalytically critical Arg-293 does not interact with the bound nucleotide. Hypothetical ring-ring stacking and peptide threading models for Rubisco reactivation are briefly discussed.
Asunto(s)
Larrea/enzimología , Modelos Moleculares , Proteínas de Plantas/química , Cristalografía por Rayos X , Interacciones Hidrofóbicas e Hidrofílicas , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
The fluorescent non-canonical amino acid (fNCAA) L-(7-hydroxycoumarin-4-yl)ethylglycine (7-HCAA) contains a photoacidic 7-hydroxycoumarin (7-HC) side chain whose fluorescence properties can be tuned by its environment. In proteins, many alterations to 7-HCAA's fluorescence spectra have been reported including increases and decreases in intensity and red- and blue-shifted emission maxima. The ability to rationally design protein environments that alter 7-HCAA's fluorescence properties in predictable ways could lead to novel protein-based sensors of biological function. However, these efforts are likely limited by a lack of structural characterization of 7-HCAA-containing proteins. Here, we report the steady-state spectroscopic and x-ray crystallographic characterization of a 7-HCAA-containing antibody fragment (in the apo and antigen-bound forms) in which a substantially blue-shifted 7-HCAA emission maximum (â¼70 nm) is observed relative to the free amino acid. Our structural characterization of these proteins provides evidence that the blue shift is a consequence of the fact that excited state proton transfer (ESPT) from the 7-HC phenol has been almost completely blocked by interactions with the protein backbone. Furthermore, a direct interaction between a residue in the antigen and the fluorophore served to further block proton transfer relative to the apoprotein. The structural basis of the unprecedented blue shift in 7-HCAA emission reported here provides a framework for the development of new fluorescent protein-based sensors.
Asunto(s)
Técnicas Biosensibles , Glicina/análogos & derivados , Fragmentos de Inmunoglobulinas , Proteínas Luminiscentes , Protones , Umbeliferonas , Cristalografía por Rayos X , Colorantes Fluorescentes/química , Glicina/química , Glicina/genética , Fragmentos de Inmunoglobulinas/química , Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , Espectrometría de Fluorescencia , Umbeliferonas/químicaRESUMEN
mKeima is an unusual monomeric red fluorescent protein (lambda(em)(max) approximately 620 nm) that is maximally excited in the blue (lambda(ex)(max) approximately 440 nm). The large Stokes shift suggests that the chromophore is normally protonated. A 1.63 A resolution structure of mKeima reveals the chromophore to be imbedded in a novel hydrogen bond network, different than in GFP, which could support proton transfer from the chromophore hydroxyl, via Ser142, to Asp157. At low temperatures the emission contains a green component (lambda(em)(max) approximately 535 nm), enhanced by deuterium substitution, presumably resulting from reduced proton transfer efficiency. Ultrafast pump/probe studies reveal a rising component in the 610 nm emission with a lifetime of approximately 4 ps, characterizing the rate of proton transfer. Mutation of Asp157 to neutral Asn changes the chromophore resting charge state to anionic (lambda(ex)(max) approximately 565 nm, lambda(em)(max) approximately 620 nm). Thus, excited state proton transfer (ESPT) explains the large Stokes shift. This work unambiguously characterizes green emission from the protonated acylimine chromophore of red fluorescent proteins.
Asunto(s)
Proteínas Luminiscentes/química , Protones , Iminas/química , Modelos Moleculares , Conformación Molecular , Solventes/química , Temperatura , Proteína Fluorescente RojaRESUMEN
Crystal structures of the photoactivatable green fluorescent protein T203H variant (PA-GFP) have been solved in the native and photoactivated states, which under 488 nm illumination are dark and brightly fluorescent, respectively. We demonstrate that photoactivation of PA-GFP is the result of a UV-induced decarboxylation of the Glu222 side chain that shifts the chromophore equilibrium to the anionic form. Coupled with the T203H mutation, which stabilizes the native PA-GFP neutral chromophore, Glu222 decarboxylation yields a 100-fold contrast enhancement relative to wild-type GFP (WT). Additionally, the structures provide insights into the spectroscopic differences between WT and PA-GFP steady-state fluorescence maxima and excited-state proton transfer dynamics.
Asunto(s)
Proteínas Fluorescentes Verdes/química , Fotoquímica/métodos , Absorción , Electrones , Proteínas Fluorescentes Verdes/metabolismo , Enlace de Hidrógeno , Luz , Modelos Químicos , Conformación Molecular , Mutación , Protones , Espectrometría de Fluorescencia/métodos , Espectrofotometría , Rayos UltravioletaRESUMEN
The arsenal of engineered variants of the GFP [green FP (fluorescent protein)] from Aequorea jellyfish provides researchers with a powerful set of tools for use in biochemical and cell biology research. The recent discovery of diverse FPs in Anthozoa coral species has provided protein engineers with an abundance of alternative progenitor FPs from which improved variants that complement or supersede existing Aequorea GFP variants could be derived. Here, we report the engineering of the first monomeric version of the tetrameric CFP (cyan FP) cFP484 from Clavularia coral. Starting from a designed synthetic gene library with mammalian codon preferences, we identified dimeric cFP484 variants with fluorescent brightness significantly greater than the wild-type protein. Following incorporation of dimer-breaking mutations and extensive directed evolution with selection for blue-shifted emission, high fluorescent brightness and photostability, we arrived at an optimized variant that we have named mTFP1 [monomeric TFP1 (teal FP 1)]. The new mTFP1 is one of the brightest and most photostable FPs reported to date. In addition, the fluorescence is insensitive to physiologically relevant pH changes and the fluorescence lifetime decay is best fitted as a single exponential. The 1.19 A crystal structure (1 A=0.1 nm) of mTFP1 confirms the monomeric structure and reveals an unusually distorted chromophore conformation. As we experimentally demonstrate, the high quantum yield of mTFP1 (0.85) makes it particularly suitable as a replacement for ECFP (enhanced CFP) or Cerulean as a FRET (fluorescence resonance energy transfer) donor to either a yellow or orange FP acceptor.
Asunto(s)
Antozoos/metabolismo , Evolución Molecular Dirigida/métodos , Proteínas Fluorescentes Verdes/metabolismo , Animales , Escherichia coli , Fluorescencia , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Células HeLa , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación ProteicaRESUMEN
pH sensing is crucial for survival of most organisms, yet the molecular basis of such sensing is poorly understood. Here, we present an atomic resolution structure of the periplasmic portion of the acid-sensing chemoreceptor, TlpB, from the gastric pathogen Helicobacter pylori. The structure reveals a universal signaling fold, a PAS domain, with a molecule of urea bound with high affinity. Through biophysical, biochemical, and in vivo mutagenesis studies, we show that urea and the urea-binding site residues play critical roles in the ability of H. pylori to sense acid. Our signaling model predicts that protonation events at Asp114, affected by changes in pH, dictate the stability of TlpB through urea binding.
Asunto(s)
Proteínas Bacterianas/química , Helicobacter pylori/metabolismo , Protones , Receptores de Superficie Celular/química , Urea/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Dimerización , Escherichia coli , Helicobacter pylori/genética , Concentración de Iones de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Plásmidos , Unión Proteica , Estabilidad Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Transducción de Señal , Urea/metabolismoRESUMEN
Fluorescent protein (FP) variants that can be reversibly converted between fluorescent and nonfluorescent states have proven to be a catalyst for innovation in the field of fluorescence microscopy. However, the structural basis of the process remains poorly understood. High-resolution structures of a FP derived from Clavularia in both the fluorescent and the light-induced nonfluorescent states reveal that the rapid and complete loss of fluorescence observed upon illumination with 450-nm light results from cis-trans isomerization of the chromophore. The photoinduced change in configuration from the well ordered cis isomer to the highly nonplanar and disordered trans isomer is accompanied by a dramatic rearrangement of internal side chains. Taken together, the structures provide an explanation for the loss of fluorescence upon illumination, the slow light-independent recovery, and the rapid light-induced recovery of fluorescence. The fundamental mechanism appears to be common to all of the photoactivatable and reversibly photoswitchable FPs reported to date.
Asunto(s)
Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/metabolismo , Fotoblanqueo , Ingeniería de Proteínas , Homología Estructural de Proteína , Animales , Antozoos , Cristalografía por Rayos X , Evolución Molecular DirigidaRESUMEN
Passive fluorescent protein markers are indispensable for dynamic cellular imaging; however, they are unselective, introduce constant background fluorescence, and require continuous observation. Photoactivatable fluorescent proteins have now been developed whose fluorescence can be switched on and off by illumination, allowing selective and direct tracking of tagged objects without the need for continuous imaging. The "kindling fluorescent protein" is a photoactivatable marker with a novel twist: it turns itself off after a selectable period.
Asunto(s)
Diagnóstico por Imagen/métodos , Fluorescencia , Proteínas Luminiscentes/química , Animales , Biomarcadores , Humanos , Microscopía Fluorescente/métodos , Fotoquímica , Factores de TiempoRESUMEN
Fluorescent proteins isolated from coral reef organisms can be roughly grouped into four color classes by emission, cyan, green, yellow, and red. To gain insight into the structural basis for cyan emission, the crystal structure of amFP486 (lambda(em)max = 486 nm) was determined by molecular replacement, and the model was refined at 1.65-A resolution. The electron density map reveals a chromophore formed from the tripeptide sequence -K-Y-G- that is indistinguishable from that of GFP (lambda(em)max = 509 nm). However, the chromophore environment closely parallels those of the yellow- and red-shifted homologs zFP538, DsRed, and eqFP611. Mutagenesis was performed for Glu-150, Ala-165, His-199, and Glu-217, which are immediately adjacent to the chromophore. His-199 and Ala-165 are key side chains responsible for the blue shift, presumably by localizing chromophore charge density on the phenolate moiety. Furthermore, in the H199T mutant the fluorescence quantum yield is reduced by a factor of approximately 110. The crystal structures of H199T (lambda(em)max = 515 nm) and E150Q (lambda(em)max = 506 nm) were determined. Remarkably, the H199T structure reveals that the stacking interaction of His-199 with the chromophore also controls the fluorescence efficiency, because the chromophore is statistically distributed in a 1:1 ratio between cis (fluorescent) and trans (nonfluorescent) conformations.
Asunto(s)
Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/metabolismo , Mutación/genética , Anémonas de Mar/química , Anémonas de Mar/genética , Animales , Cristalografía por Rayos X , Fluorescencia , Proteínas Fluorescentes Verdes/genética , Modelos Moleculares , Polietilenglicoles , Estructura Terciaria de Proteína , Análisis Espectral , Relación Estructura-ActividadRESUMEN
An in vitro system was created to study the process of membrane protein degradation by using photosystem I (PS1) as a model membrane protein. Purified chloroplast membranes were incubated at 30 degrees C in a defined buffer along with various extracts or reagents to reconstitute the disassembly and degradation of PS1, which was monitored by a variety of techniques that probe the integrity of the PS1 complex: photo-biochemical assays, semi-native gel electrophoresis, low temperature fluorescence spectroscopy, and immunoblots using antibodies against different PS1 subunits. During a typical time course, degradation of PS1 appeared to be a multievent process, with disassembly of the complex preceding proteolysis of the subunits. The first change seen was a rapid (<5 min) decrease in PS1 photochemical activity. This was followed by a diminution of far-red fluorescence emission from the core antenna of PS1 and a slower disassembly of the PS1 chlorophyll-protein core complex, as visualized by semi-native gel electrophoresis. Surprisingly, the latter was not accompanied by a similar rate of proteolysis of the PsaA core subunit. In contrast, addition of soluble proteases caused rapid loss of immuno-detectable PS1 polypeptides and cleavage of the major PS1 polypeptides in interhelical loops. The in vitro degradation process was time- and temperature-dependent but did not require ATP, GTP, or soluble chloroplast proteins. Chelation of divalent cations by EDTA inhibited the later steps of disassembly and proteolysis, and this effect could be reversed by addition of micromolar Zn2+, with Co2+ and Ca2+ providing somewhat lower activity.