RESUMO
We present a web server that predicts the far-UV circular dichroism (CD) spectra of proteins by utilizing their three-dimensional (3D) structures from the Protein Data Bank (PDB). The main algorithm is based on the classical theory of optical activity together with a set of atomic complex polarizabilities, which are obtained from the analysis of a series of synchrotron radiation CD spectra and their related 3D structures from the PDB. The results of our knowledge-based CD method (KCD) are in good agreement with measured spectra that could include the effect of D-amino acids. Our method also delivers some of the most accurate predictions, in comparison with the calculated spectra from well-established models. Specifically, using a metric of closeness based on normalized absolute deviations between experimental and calculated spectra, the mean values for a series of 57 test proteins give the following figures for such models: 0.26 KCD, 0.27 PDBMD2CD, 0.30 SESCA, and 0.47 DichroCalc. From another point of view, it is worth mentioning the remarkable capabilities of the recent approaches based on artificial intelligence, which can precisely predict the native structure of proteins. The structure of proteins, however, is flexible and can be modified by a diversity of environmental factors such as interactions with other molecules, mechanical stresses, variations of temperature, pH, or ionic strength. Experimental CD spectra together with reliable predictions can be utilized to assess eventual secondary structural changes. A similar kind of evaluation can be done for the case of an incomplete protein structure that has been reconstructed by using different approaches. The KCD method can be freely accessed from: https://kcd.cinvestav.mx/.
Assuntos
Inteligência Artificial , Proteínas , Dicroísmo Circular , Proteínas/química , Algoritmos , AminoácidosRESUMO
We present a model of circular dichroism for proteins, which is mainly based on both the classical theory of optical activity and a series of effective atomic polarizabilities. Such polarizabilities are extracted from the analysis of a set of synchrotron radiation circular dichroism spectra and their corresponding three-dimensional structures from the Protein Data Bank. Each modeled spectrum is obtained from the protein atomic coordinates and the identification of its secondary structure elements. The resulting spectra are in good agreement with additional experimental data and also with the predictions of some other models. Among them, only our approach is able to describe the effect of d-amino acids. Moreover, our model is also utilized to evaluate protein reconstructions as well as structural changes.
Assuntos
Proteínas , Síncrotrons , Dicroísmo Circular , Bases de Dados de Proteínas , Estrutura Secundária de ProteínaRESUMO
Resumen El parásito intracelular Leishmania braziliensis es el agente causal de la leishmaniasis cutánea, enfermedad endémica de zonas tropicales, cuyos tratamientos farmacológicos son tóxicos y para la cual no se dispone de una vacuna en la actualidad. Por esta razón, el estudio de las proteínas relacionadas con el metabolismo energético del parásito es relevante dada su importancia para la supervivencia del mismo. En este estudio, utilizando como secuencia plantilla los primeros 18 residuos del extremo N-terminal de la proteína nicotinamida/ nicotinato mononucleótido adenilil transferasa de L. braziliensis (Lb-NMNAT), se sintetizaron péptidos implementando la estrategia Fmoc/ tert-Butilo en una resina Rink amida MBHA. Los péptidos se purificaron por cromatografía en columna C18 y se caracterizaron mediante RP-HPLC. La proteína recombinante 6xHisLb-NMNAT se expresó en células Escherichia coli M15 y se purificó parcialmente empleando cromatografía de afinidad a metales inmovilizados. De esta proteína se confirmó su actividad enzimática a través de ensayos enzimáticos directos analizados por RP-HPLC. Los péptidos sintetizados se utilizaron para evaluar su efecto sobre la actividad enzimática de la proteína 6xHisLb-NMNAT, observándose una modulación diferencial, lo cual resulta promisorio para el diseño de herramientas quimioterapéuticas basadas en la secuencia N-terminal de la proteína Lb-NMNAT.
Abstract The intracellular parasite Leishmania braziliensis is the etiological agent of cutaneous leishmaniasis, an endemic disease in the tropics, whose pharmacological treatments are toxic and for which there is currently no vaccine. For this reason, the study of proteins related to the energy metabolism of the parasite is relevant given its importance for its survival. In this study, based on the first 18 residues of the N-terminal end of the nicotinamide/nicotinate mononucleotide adenylyl transferase protein from L. braziliensis (Lb-NMNAT) as a template, peptides were synthesized implementing the Fmoc/tert-Butyl strategy in a Rink amide MBHA resin. The peptides were purified by C18 column chromatography and characterized by RP-HPLC. The recombinant 6xHisLb-NMNAT protein was expressed in Escherichia coli M15 cells and partially purified using immobilized metal affinity chromatography. The enzymatic activity of the protein was confirmed through direct enzymatic assays analyzed by RP-HPLC. The synthesized peptides were used to evaluate their effect on the enzymatic activity of the 6xHisLb-NMNAT protein, observing a differential modulation, which is promising for the design of chemotherapeutic tools based on the N-terminal sequence of the Lb-NMNAT protein.
Resumo O parasita intracelular Leishmania braziliensis é o agente causador da leishmaniose tegumentar, doença endêmica nos trópicos, cujos tratamentos farmacológicos são tóxicos e para a qual não existe vacina atualmente. Por este motivo, o estudo de proteínas relacionadas ao metabolismo energético do parasita é relevante dada a sua importância para a sua sobrevivência. Neste estudo, usando os primeiros 18 resíduos da extremidade N-terminal da proteína adenilil transferase de nicotinamida/mononucleotídeo nicotinato de L. braziliensis (Lb-NMNAT) como uma sequência modelo, os peptídeos foram sintetizados implementando a estratégia Fmoc/tert-Butil em uma resina Rink amida MBHA. Os peptídeos foram purificados por cromatografia em coluna C18 e caracterizados por RP-HPLC. A proteína recombinante 6xHisLb-NMNAT foi expressa em células de Escherichia coli M15 e parcialmente purificada usando cromatografia de afinidade com metal imobilizado. Desta proteína, sua atividade enzimática foi confirmada por meio de ensaios enzimáticos diretos analisados por RP-HPLC. Os peptídeos sintetizados foram utilizados para avaliar seu efeito na atividade enzimática da proteína 6xHisLb-NMNAT, observando uma modulação diferencial, o que é promissor para o projeto de ferramentas quimioterápicas baseadas na sequência N-terminal da proteína Lb-NMNAT
RESUMO
We present a model of circular dichroism for proteins that is based on the classical electromagnetic theory for optical activity. The two additional constituents of the model are as follows: an appropriate characterization of the secondary structure of the protein residues and the assignment of an effective polarizability to each type of classified residue. The set of effective polarizabilities is obtained by means of a Monte Carlo statistical method, which is used to analyze a series of synchrotron radiation circular dichroism spectra together with their corresponding crystallographic structures. As a result, the predicted spectra from our model are in good accord with experimental data, as well as with the results of some other theoretical approaches.