3.2. Probes: Fluorescence, Fluorescence Resonant Energy Transfer (FRET), Spin Probes

One of the main needs in studying dynamic systems like muscles is to be able to probe specific molecules, for example the myosin head, or myosin binding protein-C (MBP-C), or domains of actin, or troponin and tropomyosin, in such a way as to determine their orientation and/or their mobility under different physiological conditions. Later we will see that some of this can be done by X-ray diffraction, but here we consider the unique contribution that spectroscopy can make. We start with fluorescence. It happens that amino acids like tryptophan are intrinsically fluorescent [100,101,102]. When tryptophan is illuminated by incident radiation of appropriate wavelength (around 3000 Å) electrons in the ground state (level S₀; Figure 14) are excited to higher energy levels (e.g., upper levels in state S₁) and then, after some non-radiative jumps between vibration levels within state S₁, they jump back down to the ground state and re-emit radiation at a lower energy (longer wavelength). If the incident light is polarised, in the case of muscle either parallel (Ppar, intensity Ipar) or perpendicular (Pperp, intensity Iperp) to the fibre axis, then the polarization of the emitted fluorescence is defined as P = (Ipar − Iperp)/(Ipar + Iperp) which is sensitive to the orientation of the fluorophore. It was shown by Aronson and Morales [102] that P is sensitive to the physiological state of the muscle, relaxed, rigor or active, and, although there are tryptophans in other molecules than myosin, Nihei et al. [103] showed that most of the signal was actually coming from the myosin heads.

Probes: (a) Energy levels in fluorescence probes, (b) A: EPR: Spin up and spin down (M_s = ± 1/2) energy levels in a magnetic field B_0, which become more widely separated the stronger the field_, and a microwave source on the right that can flip the spins. Inset (B), the microwave absorption spectrum (top) from the spin flip in A and its first derivative (below). For details see text.

To be more specific, and to label other things than the myosin head, extrinsic fluorophores can be added to skinned fibres (fibres with their outer membrane (sarcolemma) removed either physically or chemically using detergents) in which some of the known molecules, such as troponin or the myosin light chains or MyBP-C, can be exchanged for equivalent purified proteins carrying extrinsic fluorescent tags, such as IAEDANS [71]. One of the problems of such extrinsic fluorophores is that they can have significant mobility on their parent molecule, even if the molecule itself is fairly static. This mobility can be reduced substantially by the use of bi-functional probes, where the probe is covalently linked to two sites on the parent molecule rather than one (e.g., BR, bifunctional rhodamine [104]).

Fluorescence can also be used to determine distances if there is a donor fluorophore and a receptor within about 100 Å [105,106]. This makes use of what is known as Forster resonance energy transfer (FRET, sometimes called fluorescence resonance energy transfer). This can either use intrinsic fluorophores like tryptophan or added fluorescent labels with mono- or bi-functional binding [105,106]. A possible problem with extrinsic labels is that they may modify the way that a protein normally functions. It is important in the case of muscle to test whether force generation and movement are affected by the labelling.

Exciting developments in spectroscopic methods include luminescence resonance energy transfer (LRET) and total internal reflection fluorescence detection (TIRF [107]). Despite the limited resolution, the latter can ‘detect’ fluorescence from labels on single molecules. Special Issue reference [108] discusses the new technique of ‘time-resolved’ fluorescence resonance energy transfer (TR-FRET) which can directly resolve structural states in the strongly-bound actin-myosin interaction.

A complementary method to using fluorescence is to use spin probes [109]. The technique, known as electron paramagnetic resonance (EPR) or electron spin resonance (ESR), makes use of the fact that all electrons possess a magnetic moment and will align in a magnetic field. If there is an unpaired electron, this will align either parallel to or antiparallel to the field. The antiparallel configuration (M_s = +½) has slightly more energy than the parallel alignment (M_s = −½: Figure 14b). A common arrangement is to have the sample, which may be a solution or a skinned or intact fibre, in a strong magnetic field. Electrons are promoted from the lower level (Figure 14b: spin −½) to the upper level (spin +½) by an incident microwave beam of appropriate frequency (energy). With the microwave frequency scanned past the appropriate excitation frequency, the absorbtion spectrum can be detected, recorded (Figure 14b; Panel B) and its first derivative calculated. Alternatively, this can also be done the other way round with the microwave source of fixed frequency and the magnetic field scanned through the optimal field strength.

As with fluorescence, the spin label can be an extrinsic probe such as a nitroxide radical (site directed spin labelling), or it can also be a bifunctional probe to reduce probe mobility, but once again there is a need to check that the probe is not altering the normal function of the host protein. The precise application of EPR depends both on the field strength, the excitation frequency and which signal is recorded. Conventional EPR uses fields of about 3500 Gauss and frequencies in the range 9-10 GHz (X-band). With this set-up, motions with very fast correlation times can be detected (~10⁻⁹ to 10⁻⁷ s) using conventional absorbtion. However, muscle proteins have interesting motions in the microsecond to millisecond time window. This window can be studied using saturation transfer EPR (ST-EPR; [109,110,111]). EPR can be used to measure both motions and probe orientations. In addition, measurements of distance can be determined by double electron-electron resonance (DEER) and ab initio high resolution structure determined in muscle fibres by a combination of two-probe BEER with single probe EPR [108,109,110,111]. Bifunctional probes attached to the lever arm of the myosin head can be used to determine the lever arm orientation [111].