SPETTROFLUORIMETRIA: FLORESCENZA INTRINSECA Stefania Iametti DeFENS Università degli Studi di Milano La fluorescenza sfrutta la capacità di alcune specie chimiche di riemettere in un salto quantico discreto e ampio l’energia assorbita sottoforma di radiazione luminosa Non tutta l’energia assorbita viene ri-emessa (resa quantica), e la lunghezza d’onda di emissione è per forza superiore (minor frequenza) rispetto a quella di eccitazione FLUORIMETRO SPETTROFLUORIMETRIA Caratteristiche della fluorescenza di alcuni residui aminoacidici Fluorescenza intrinseca Fluorescenza dipende ……. dal SOLVENTE ……..dal pH … dalla forza ionica A schematic view of changes in tryptophan sidechain exposure upon structural modification of proteins buried Trp, blue-shifted fluorescence, low emission buried Trp, blue-shifted fluorescence, high emission solvent-exposed Trp, red-shifted fluorescence, high emission “quenching” residue NATIVE PARTIALLY UNFOLDED FULLY UNFOLDED Posizione deiTRP nella struttura nativa ………posizione TRP nell’organizzazione strutturale: Distinguere tra diverse organizzazioni strutturali ………posizione TRP nell’organizzazione strutturale: Distinguere tra diversi stati di “folding” e “unfolding” ………posizione TRP nell’organizzazione strutturale Distinguere tra diversi stati strutturali: Associazione tra proteine ………posizione TRP nell’organizzazione strutturale Sensibilità agli agenti “quenchanti” Nessun effetto: Trp “sepolto” e inaccessibile Marcato effetto: Trp vicino alla superficie Analizzare gli effetti “quenchanti”: Il grafico di Stern-Vollmer CASE 1 Structural modification: ALA BLG Buried Trps Exposed Trp Carboxylatecoordinate, structurestabilizing Ca++ ion OFF-LINE APPLICATIONS: Ca++ and temperature dependent changes in alpha-lactalbumin Trp fluorescence “assenza Ca++” “presenza Ca++” L.EYNARD, S.IAMETTI, P.RELKIN, F.BONOMI. Surface hydrophobicity changes and heat-induced modifications of alphalactalbumin. J. Agric. Food Chem., 40 (1992) 1731-1736 Hydrophobic sites in betalactoglobulin, “telltale” residues, “silent” residues, patches and quilts large central hydrophobic cavity hydrophobic surface groove Trp 19 Trp 61 hydrophobic surface regions involved in dimer contact DENATURAZIONE CHIMICA Urea 0M Urea 1M Urea 2M Urea 3M Urea 4M Urea 4.5M Urea 5M Urea 5.5M Urea 6M Urea 6.5M Urea 7M Urea 7.5M Urea 8M 150 100 Urea 0M Urea 1M Urea 2M Urea 3M Urea 4M Urea 4.5M Urea 5M Urea 5.5M Urea 6M Urea 6.5M Urea 7M Urea 7.5M Urea 8M 200 Fluorescenza, A.U. Fluorescenza, A.U. 200 150 100 50 50 proteina proteina + ligando 0 0 300 320 340 360 380 400 420 300 440 320 340 360 380 400 420 440 Lunghezza d'onda, nm Lunghezza d'onda, nm 220 360 Fluorescenza, A.U. 200 λ al massimo di fluorescenza, nm proteinaBLG BLG -palmitato proteina + ligando 180 160 proteina BLG BLG-palmitato proteina + ligando 355 350 345 340 140 335 0 2 4 [urea], M 6 8 0 2 4 [urea], M 6 8 Controlling the polymers formation polymers of BLG: - stem from structurally modified monomers - may be stabilized by various kinds of bonds - may have different properties (geometric & physical) may get really large! Controlling the polymers formation chaotrope-dependent features of partially unfolded intermediates (1): structural signatures urea KSCN denaturazione fisica progressive heating of beta-lactoglobulin: effects of the aggregation state D. FESSAS, S. IAMETTI, A. SCHIRALDI, F. BONOMI. Thermal unfolding of monomeric and dimeric βlactoglobulins. Eur. J. Biochem. (2001) 268, 5439-5448 CASO 2: DENATURAZIONE INTERFACCIALE DI PROTEINE: ruolo di interfacce solide e liquide e la denaturazione di BLG Emulsione: la proteina penetra la fase apolare Adsorbimento: la proteina non penetra la fase solida TRP FLUORESCENCE TO ADDRESS CHANGES IN TERTIARY STRUCTURE BLG on oil droplets BLG on NP 400 160 NP + β-lactoglobulin β-lactoglobulin Fluorescence Intensity (A.U.) Fluorescence Intensity (A.U.) Fluorescence Intensity (A.U.) 140 120 100 80 60 40 native 20 300 200 native 100 0 -20 300 0 310 320 330 340 350 360 Wavelength (nm) 370 380 390 400 300 325 350 375 400 425 450 475 500 Wavelength (nm) When BLG is adsorbed on the surface of the oil droplets, the Trp19 red shift is slightly higher compared to NP-adsorbed BLG. Moreover, Trp in NP-adsorbed BLG shows a ≈2 folds higher quantum yield. Spiegare le variazioni in intensità: Cys66-Cys160 Trp61 Trp19 Changes in quantum yield could be explained by the moving of Trp residues away from quenchers: Trp61 away from the disulphide bond Cys66-Cys160, and/or Trp19 away from Arg124. Arg124 BLG interactions 1: solids in foods Trp fluorescence NP-bound free Cys 121 reactivity Unfolding of BLG may be monitored by measuring exposure of individual sidechains,such as Trp19 or Cys121… 2 – Conditional functionality The large structural changes in NP-bound BLG do not prevent binding of hydrophobes, an issue of practical relevance in drug and nutrient delivery L’approccio funziona anche con altre proteine: Il caso della soia emulsion Urea denatured native Le proteine strutturalmente modificate in emulsioni modello sono solo quelle legate alla fase lipidica Gli spettri di emissione sono stati registrati sull’emulsione e sulle fasi separate per centrifugazione mixture aqueous phase Lipidbound Insoluble proteins are generated in many food processes... Cheese making CLOT RAW MILK heat PASTEURIZED MILK enzymes CHEESE enzymes WHEY heat RICOTTA SEMOLINA kneading DOUGH drying RAW PASTA Pasta making (& cooking) boiling COOKED PASTA ...or may be present since the very beginning (the more, the better) The general problem: Addressing structural changes in waterinsoluble proteins Found in many food systems Vegetable “storage” proteins Soluble in acids or bases glutelins alchools (EtOH, PrOH) prolamines little structure is retained after their solubilization in non aqueous solvents, acids/bases or denaturing buffers! Some particular problems: 1 - understand structural differences among very similar proteins: performance related? These proteins are the main contributor to the structure of the product 2 - monitor conformational changes ensuing from treatments (solvation & mechanical treatment) Treatment-induced modification establish the nature of the product 3 - follow phase transfer phenomena Water transfer: protein to starch ⇒ bread cooking starch to protein ⇒ bread staling SOLID STATE SPECTROSCOPIES What are they? spectroscopic tools for the investigation of solids/gels without resorting to the extraction/solubilization of individual components (with concomitant loss of structure!) IR & NIR NMR Fluorescence What for? Analytical purposes Structural studies on individual components on their interaction A possible solution: Solid-state spectroscopy: fluorescence Fluorescence provides evidence on the chemical environment around “fluorescent probes”, either embedded in the protein structure (e.g., Trp residues) or capable to bind to specific protein regions. Thus, properties of the protein structure, and their changes during processes may be monitored under “non-destructive” conditions. FRONT-FACE FLUORESCENCE FOR PROTEIN STRUCTURAL STUDIES ON SOLID SAMPLES excitation beam emitted fluorescence, to emission monochromator reflected light, discarded solid sample quartz window mechanical support standard cell TRYPTOPHAN FRONT-FACE FLUORESCENCE DETECTS DIFFERENT SOLVATION OF PROTEINS IN DOUGH 380 emission maximum, nm fluorescence intensity, a. u. 150 100 50 370 360 350 10 20 30 40 50 water content, % 0 350 400 wavelength, nm 450 BONOMI F., MORA G., PAGANI M. A., IAMETTI S. Probing the structural features and the solvation behaviour of wheat proteins by front-face fluorescence. Anal. Biochem. (2004) 329, 10411 Solid-state fluorometry Solvation of wheat proteins Protein solvation is the earliest, necessary step in all processes that involve flour (of all kinds) Addition of water transforms the higly dehydrated, compact protein structures in the original dry kernel into fluffier and more open structures Solid-state fluorometry Not all proteins were created equal Solvation behavior differs in soft and hard wheat, and within varieties Intrinsic fluorescence (tryptophan) allows to discriminate between various wheat-based starting materials and to detect their different solvation behaviour emission maximum, nm 380 370 360 flour 350 semolina 10 20 30 40 water content, % 50 5 - … O ADATTARE QUEL CHE C’E’ Spectroscopic approaches have been modified to suite the physical nature of the samples: -solid-state fluorescence of spaghetti A BIG “THANK YOU” Rest of the world The DeFENS Team BLG unfolding Pasquale Ferranti (Naples) Ivano Eberini (UniMi) Stefania Iametti Alberto Barbiroli Mauro Marengo Matteo Miriani Emulsions & doughs Milena Corredig (UGuelph) Koushik Seetharaman† Ambogina Pagani Alessandra Marti Patrizia Rasmussen (& many students)
© Copyright 2024 Paperzz
