Energy transfer between terbium (III) and cobalt (II) in thermolysin: a new class of metal--metal distance probes.

Abstract

The visible fluorescence of terbium(III) when bound to a calcium binding site of thermolysin is greatly enhanced with an excitation maximum at 280 nm but substitution of cobalt(II) for zinc at the-active site decreases the intensity by 89.5%. Treatment with N-bromosuccinimide quenches enzyme tryptophan and Tb(III) fluorescence to a similar extent and suggests the operation of tryptophan Tb(III) -Co(II) energy relay system in the enzyme. Dipoledipole radiationless energy transfer between the Tb(III) donor and the Co(II) acceptor can account for this quenching. The inherent characteristics of the metal pair limits the value of the orientation factor, K2, of the Fbrster equation, thereby reducing uncertainties in distance measurements by energy transfer compared with other systems. A quantum yield of 0.51 yields a value of Bo, the distance for 50% energy transfer, of 19.6 A, and a distance, R, between Tb(III) and Co(II) of 13.7 A, a value identical to that measured for the distance between the active site zinc atom and calcium atom number 1 by x-ray analysis in native thermolysin crystals.The limits of confidence of this measurement are discussed.Energy transfer between two different metal atom sites of a protein provides a new class of probes to measure intramolecular distances of biological macromolecules in solution.The substitution of paramagnetic, chromophoric metal ions, e.g., Co(II), Cu(II), or trivalent lanthanides, for spectroscopically silent ions which are native to metalloenzymes, e.g., Ca(II), Mg(II), or Zn(II), has proven increasingly useful in the study of their structure-function relationships (1). In the arsenal of spectroscopic techniques, fluorescence measurements offer particular advantages, since they can be employed in the study of biological molecules at relatively low concentrations. Certain of the lanthanide ions, principally Tb(III) and Eu(III), can exhibit considerably enhanced fluorescence when replacing Ca(II) in calcium-dependent proteins (2-4) or when bound to nucleic acids (5). Radiationless energy transfer is now an established means to probe distance relationships between organic fluorophoric donors and organic chromophoric acceptors (6, 7).We have now monitored energy transfer between metal ion donors and metal ion acceptors and thereby measured distances between different metal ion sites of thermolysin, a metalloendopeptidase whose metal content and three-dimensional structure are known (8,9 (Fig. 1). * To whom correspondence should be addressed.Hence, Co(II) and Tb(III) substituted thermolysin favors obh servation of inter-ion energy transfer and, owing to its known structure, provides a suitable benchmark for future distance determinations using this technique. EXPERIMENTAL Thermolysin, obtained from Calbiochem (San Diego, Calif.), was recrystallized and rendered zinc free as described (11). Enzyme concentrations were determined by measuring the absorbance at 280 nm, based on the reported absorbance E1%280 = 17.65 (14) and a molecular weight of 34,600 (8).The enzyme was...

Characterization of powellite-based solid solutions by site-selective time resolved laser fluorescence spectroscopy

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Short-distance probes for protein backbone structure based on energy transfer between bimane and transition metal ions

“…This technique should open the door to the complete mapping of unknown protein structures or changes in protein structure with FRET. (12)(13)(14)(15). The absorbance spectra of these metals are broad, and their extinction coefficients are very small, between 5-and 30-fold less than commonly used organic fluorophores (12).…”

Transition metal ion FRET to measure short-range distances at the intracellular surface of the plasma membrane

“…The FRET efficiency is steeply dependent on distance between the donor and acceptor, allowing tmFRET to serve as a molecular ruler for distances in the membrane. The main advantages of tmFRET over classical FRET methods are (a) R 0 is very short (10-20 Å), allowing short-range interactions to be studied; (b) the transition metals have multiple transition dipoles, reducing the orientation dependence of FRET (Horrocks et al, 1975); (c) the metals can be reversibly bound to native and introduced binding sites; and (d) different metals have different absorption properties, and therefore R 0 's, allowing the choice of metal to be tuned to the distance of interest.Extending tmFRET to studying dynamics in cell membranes would provide a powerful tool for exploring the structure of the membrane (e.g., microdomains), assembly of multiprotein complexes (e.g., scaffolding proteins and their associated proteins), rearrangements Transition metal ion FRET to measure short-range distances at the intracellular surface of the plasma membrane Biological membranes are complex assemblies of lipids and proteins that serve as platforms for cell signaling. We have developed a novel method for measuring the structure and dynamics of the membrane based on fluorescence resonance energy transfer (FRET).…”

“…The FRET efficiency is steeply dependent on distance between the donor and acceptor, allowing tmFRET to serve as a molecular ruler for distances in the membrane. The main advantages of tmFRET over classical FRET methods are (a) R 0 is very short (10-20 Å), allowing short-range interactions to be studied; (b) the transition metals have multiple transition dipoles, reducing the orientation dependence of FRET (Horrocks et al, 1975); (c) the metals can be reversibly bound to native and introduced binding sites; and (d) different metals have different absorption properties, and therefore R 0 's, allowing the choice of metal to be tuned to the distance of interest.…”

“…e d u ; or William N. Zagotta: z a g o t t a @ u w . e d u Abbreviations used in this paper: FRET, fluorescence resonance energy transfer; PCF, patch-clamp fluorometry; TIRF, total internal reflection fluorescence; tmFRET, transition metal ion FRET.donor and a nonfluorescent transition metal ion as an acceptor (Latt et al, 1970(Latt et al, , 1972Horrocks et al, 1975;Richmond et al, 2000;Sandtner et al, 2007; Taraska et al, 2009a,b;Yu et al, 2013). Transition metal ions such as Ni 2+ , Co 2+ , and Cu 2+ have broad absorption spectra that overlap the emission spectra of visible light fluorophores (see Miessler and Tarr [1999] pages 361-380 for a discussion of electronic spectra of coordination compounds).…”