El muestreo reducido no-uniforme aplicado en los experimentos 3D heteronucleares HNCO, HN(CA)CO, HNCACB y CBCA(CO)NH, redujo el tiempo de adquisición y mostró aumentar significativamente la resolución de los picos, a la vez que se disminuyó notoriamente el tamaño de los artefactos.
Mediante el conjunto de experimentos adquiridos se logró una asignación muy completa de los núcleos del sistema As-p18–ácido oleico. Esto, a su vez, permitió establecer un gran número de restricciones experimentales.
Durante el refinamiento de la estructura, la inclusión de las restricciones de puente de hidrógeno y de RDC tuvo un impacto apreciable en la dispersión del ensemble. Así, la incorporación de estos dos sets de restricciones experimentales significó una mejora en la calidad del conjunto de estructuras, con un decremento del valor de RMSD de 0.941 Å a 0.707 Å para las regiones definidas (Ver imagen Apéndice G).
El conjunto de estrategias operativas, incluyendo los experimentos de filtro isotópico y los esquemas de marcación, permitieron determinar de manera precisa la posición del ligando dentro de la proteína. Este enfoque resulta novedoso y no convencional para la determinación por RMN de estructuras de proteínas FABPs en su forma holo. En los casos hasta el momento reportados se recurre al docking del ligando sobre una estructura previamente resuelta de la proteína, o al uso de coordenadas cristalográficas en conjunto con restricciones experimentales por RMN (Lücke et al., 2006; Cai et al., 2012; Lassen et al., 1995; He et al., 2007; Eliseo et al., 2007; Tomaselli et al., 2012). El cálculo simultáneo de las dos moléculas solo se ha mencionado en la aplicación de un algoritmo de geometría de distancia por el grupo de Cistola en el año 1996 (Hodsdon et al., 1996).
Los parámetros recabados durante la validación permitieron corroborar la confiabilidad de los datos experimentales y la alta calidad del conjunto de estructuras derivado de esta información.
4.12 Bibliografía
Breeze AL. (2000) Isotope-filtered NMR methods for the study of biomolecular structure and interactions. Prog Nucl Magn Reson Spectrosc 36(4): 323-372 Brünger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J, Nilges M, Pannu NS, Read RJ, Rice LM, Simonson T, Warren GL. (1998) Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr 54(Pt 5):905-21.
Brutscher B, Schanda P. (2009) Rapid multidimensional NMR: fast-pulsing techniques and their applications to proteins. Encyclopedia of Magnetic Resonance 43: 1-10.
Cai J, Lücke C, Chen Z, Qiao Y, Klimtchuk E, Hamilton JA. (2012) Solution structure and backbone dynamics of human liver fatty acid binding protein: fatty acid binding revisited. Biophys J 102(11):2585- 94.
Cavanagh J, Fairbrother WJ, Palmer III AG, Skelton NJ. (1996) Protein NMR Spectroscopy. Principles and Practice, Academic Press, New York.
Chen J, Nietlispach D, Shaka AJ, Mandelshtam VA. (2004) Ultra-high resolution 3D NMR spectra from limited-size data sets. J Magn Reson 169(2):215-24. Chen K, Tjandra N. (2012) The use of residual dipolar coupling in studying proteins by NMR. Top Curr Chem 326:47-67.
Cheung MS, Maguire ML, Stevens TJ, Broadhurst RW. (2010) DANGLE: a Bayesian inferential method for predicting protein backbone dihedral angles and secondary structure. J Magn Reson 202(2):223–233. Cierpicki T, Bushweller JH. (2004) Charged gels as orienting media for measurement of residual dipolar couplings in soluble and integral membrane proteins. J Am Chem Soc 15;126(49):16259-66.
Clubb RT, Thanabal V, Wagner G. (1992) A constant-time three-dimensional triple-resonance pulse scheme to correlate intraresidue 1HN, 15N, and 13C' chemical shifts in 15N-13C labeled proteins. J
Magn Res (1969) 97: 213-217.
Eliseo T, Ragona L, Catalano M, Assfalg M, Paci M, Zetta L, Molinari H, Cicero DO. (2007) Structural and dynamic determinants of ligand binding in the ternary complex of chicken liver bile acid binding protein with two bile salts revealed by NMR. Biochemistry 46(44):12557-67.
Gronenborn AM. (2002) The importance of being ordered: improving NMR structures using residual dipolar couplings. C R Biol 325(9):957-66.
Grzesiek S, Bax A. (1993) Amino acid type determination in the sequential assignment procedure of uniformly 13C/15N-enriched proteins. J
Biomol NMR 3: 185-204.
Habeck M, Rieping W, Linge JP, Nilges M. (2004) NOE assignment with ARIA 2.0 protein NMR techniques. Humana Press. pp. 379-402. Humana Press. pp. 379-402.
Hansen MR, Mueller L, Pardi A. (1998) Tunable alignment of macromolecules by filamentous phage yields dipolar coupling interactions. Nat Struct Mol Biol 5: 1065-1074.
He Y, Yang X, Wang H, Estephan R, Francis F, Kodukula S, Storch J, Stark RE. (2007). Solution- state molecular structure of apo and oleate-liganded liver fatty acid-binding protein. Biochemistry 46(44):12543-56.
Hodsdon ME, Ponder JW, Cistola DP. (1996) The NMR solution structure of intestinal fatty acid-binding protein complexed with palmitate: application of a novel distance geometry algorithm. J Mol Biol 264(3):585-602.
Hoch JC, Stern AS. (2001) Maximum entropy reconstruction, spectrum analysis and deconvolution in multidimensional nuclear magnetic resonance. Methods Enzymol 338:159-78.
Hoch JC, Maciejewski MW, Mobli M, Scuyler AD, Stern AS. (2012) nonuniform sampling in multidimensional NMR. eMagRes 1: 181–196. Hu H, De Angelis AA, Mandelshtam VA, Shaka AJ. (2000) The multidimensional filter diagonalization method. J Magn Reson 144(2):357-66.
Hyberts SG, Arthanari H, Wagner G. (2012) Applications of non-uniform sampling and processing. Top Curr Chem 316:125-48.
Iwahara JJ, Wojciak M, Clubb RT. (2001) Improved NMR spectra of a protein-DNA complex through rational mutagenesis and the application of a sensitivity optimized isotope-filtered NOESY experiment. J Biomol NMR 19:231-241.
Kay LE, Xu GY, Singer AU, Muhandiram DR, Formankay JD. (1993) A gradient enhanced HCCH- TOCSY experiment for recording side-chain 1H and
13C correlations in H2O samples of proteins. J Magn
Reson (B) 101: 333-337
Kazimierczuk K, Koźmiński W, Zhukov I. (2006) Two- dimensional Fourier transform of arbitrarily sampled NMR data sets. J Magn Reson 179(2):323-8. Kirkpatrick S, Gelatt CD Jr, Vecchi MP. (1983) Optimization by simulated annealing. Science 220(4598):671-80.
Laskowski RA, Rullmann JAC, MacArthur MW, Kaptein R, Thornton JM. (1996) AQUA and PROCHECK-NMR: Programs for checking the quality of protein structures solved by NMR. J Biomol NMR 8: 477-486.
Lassen D, Lücke C, Kveder M, Mesgarzadeh A, Schmidt JM, Specht B, Lezius A, Spener F, Rüterjans H. (1995) Three-dimensional structure of bovine heart fatty-acid-binding protein with bound palmitic acid, determined by multidimensional NMR spectroscopy. Eur J Biochem 230(1):266-80. Linge JP, Nilges M (1999) Influence of non-bonded parameters on the quality of NMR structures: A new force field for NMR structure calculation. J Biomol NMR 13:51-59.
Linge JP, Williams MA, Spronk CA, Bonvin AM, Nilges M. (2003) Refinement of protein structures in explicit solvent. Proteins 20, 496–506.
Lipsitz RS, Tjandra N. (2004) Residual dipolar couplings in NMR structure analysis. Annu Rev Biophys Biomol Struct. 33:387-413.
Luan T, Jaravine V, Yee A, Arrowsmith CH, Orekhov VY. (2005) Optimization of resolution and sensitivity of 4D NOESY using multi-dimensional decomposition. J Biomol NMR 33(1):1-14.
Lücke C, Qiao Y, van Moerkerk HT, Veerkamp JH, Hamilton JA. (2006). Fatty-acid-binding protein from the flight muscle of Locusta migratoria: evolutionary variations in fatty acid binding. Biochemistry 45(20):6296-305.
Marion D. (2005) Fast acquisition of NMR spectra using Fourier transform of non-equispaced data. J Biomol NMR 32(2):141-50.
Morris AL, MacArthur MW, Hutchinson EG, Thornton JM. (1992) Stereochemical quality of protein structure coordinates. Proteins 12(4):345-64.
Moseley HN, Sahota G, Montelione GT. (2004) Assignment validation software suite for the evaluation and presentation of protein resonance assignment data. J Biomol NMR 28(4):341-55.
Nabuurs SB, Spronk CAEM, Vriend G, Vuister GW. (2004) Concepts and tools for NMR restraint analysis and validation. Concepts Magn Reson Part A 22A: 90-105.
Nilges M. (1995) Calculation of protein structures with ambiguous distance restraints. Automated assignment of ambiguous NOE crosspeaks and disulphide connectivities. J Mol Biol 245: 645-660. Ogura K, Terasawa H, Inagaki F. (1996) An improved double-tuned and isotope-filtered pulse scheme based on a pulsed field gradient and a wide-band inversion shaped pulse. J Biomol NMR 8:492-498. Orekhov VY, Ibraghimov I, Billeter M. (2003) Optimizing resolution in multidimensional NMR by three-way decomposition. J Biomol NMR 27(2):165- 73.
Ottiger M, Delaglio F, Bax A. (1998) Measurement of J and Dipolar couplings from simplified two- dimensional NMR spectra. J Magn Reson 131: 373- 378.
The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC.
Rieping W, Habeck M, Bardiaux B, Bernard A, Malliavin TE, Nilges M. (2007) ARIA2: automated NOE assignment and data integration in NMR structure calculation. Bioinformatics 23 381-382. Rovnyak D, Filip C, Itin B, Stern AS, Wagner G, Griffin RG, Hoch JC. (2003) Multiple-quantum magic- angle spinning spectroscopy using nonlinear sampling. J Magn Reson 161(1):43-55.
Rovnyak D, Frueh DP, Sastry M, Sun Z-YJ, Stern AS, Hoch JC, Wagner G. (2004) Accelerated acquisition of high resolution triple-resonance spectra using non-uniform sampling and maximum entropy reconstruction. J Magn Reson 170: 15-21.
Sacchettini JC, Gordon JI, Banaszak LJ. (1989) Crystal structure of rat intestinal fatty-acid-binding protein. Refinement and analysis of the Escherichia coli-derived protein with bound palmitate. J Mol Biol 208, 327-339
Sibisi S, Skilling J, Brereton RG, Laue ED, Staunton J. (1984) Maximum entropy signal processing in practical NMR spectroscopy. Nature 311: 446-447. Sklenar V, Piotto M, Leppik R, Saudek V. (1993) Gradient-tailored water suppression for 1H-15N HSQC
experiments optimized to retain full sensitivity. J Magn Reson Series A 102: 241-245.
Spronk C, Nabuurs S, Krieger E, Vriend G, Vuister G. (2004) Validation of protein structures derived by
NMR spectroscopy. Prog Nucl Magn Reson Spectrosc 45: 315-337.
Tycko R, Blanco FJ , Ishii Y. (2000) Alignment of biopolymers in strained gels: A new way to create detectable dipole-dipole couplings in high-resolution biomolecular NMR. J Am Chem Soc 122, 9340-9341. Tjandra N, Omichinski JG, Gronenborn AM, Clore GM, Bax A. (1997). Use of dipolar 1H-15N and 1H-13C
couplings in the structure determination of magnetically oriented macromolecules in solution. Nat Struct Biol 4:732-8.
Tomaselli S, Assfalg M, Pagano K, Cogliati C, Zanzoni S, Molinari H, Ragona L. (2012) A disulfide bridge allows for site-selective binding in liver bile acid binding protein thereby stabilising the orientation of key amino acid side chains. Chemistry 18(10):2857-66.
Vranken WF, Boucher W, Stevens TJ, Fogh RH, Pajon A, Llinas M, Ulrich EL, Markley JL, Ionides J, Laue ED. (2005) The CCPN Data Model for NMR
Spectroscopy: Developement of a Software Pipeline. Proteins 59: 687-696.
Vuister GW, Bax A. (1992) Resolution Enhancement and Spectral Editing of Uniformly 13C Enriched
Proteins by Homonuclear Broadband 13C Decoupling.
J Magn Reson 98: 428-435.
Wishart DS, Sykes BD. (1994) The 13C chemical-shift
index: a simple method for the identification of protein secondary structure using 13C chemical-shift data. J
Biomol NMR 4:171–180.
Wittekind M, Mueller L. (1993) HNCACB, a high- sensitivity 3D NMR experiment to correlate amide- proton and nitrogen resonances with the alpha- and beta-carbon resonances in proteins. J Magn Res Series B 101: 201-205.
Yamazaki T, Forman-Kay JD, Kay LE. (1993) Two- dimensional NMR experiments for correlating carbon- 13.beta. and proton.delta./.epsilon. chemical shifts of aromatic residues in 13C-labeled proteins via scalar couplings. J Am Chem Soc 115: 11054-11055.