Molecular probes for biological functions

The aim of this thesis was to develop the use of fluorescent probes for studying structural changes in biochemical systems. The investigation was carried out in two major ways:-
chromophoric molecules with different structures and fluorescence properties were used in an attempt to discover the structural characteristics needed to make a fluorescent molecule behave as a probe.
biochemical systems of differing complexity were studied using ANS (1-anilino-naphthalene-8-sulphonate) and other probes to see what sort of information this probe technique could give. The two main biochemical systems used were:- (i) the ligand induced conformational change of glutamate dehydrogenase (GDH) and, (ii) energy dependent structural changes in submitochondrial particles (SMP). (i.e. those changes induced in mitochondrial membrane fragments by oxidisable substrate which could be reversed by uncoupling agents).
a) MNS (2-(N-methyl anilino)-naphthalene-6-sulphonate) and MNS-Cl were synthesised in an attempt to label GDH covalently with an ANS type molecule. MNS was shown to behave in the same way as ANS, but with a larger blue shift of fluorescence on binding, and a larger enhancement accompanying the conformational change (3 fold instead of 2 fold). MNS-Cl denatured the enzyme even under mild conditions of modification.
A variety of dansyl (l-dimethylamino-naphthalene-5-sulphonyl) amino acids were used to probe the conformational change of GDH. The dansyl derivatives of glutamate and inhibitors of the enzyme had their fluorescence quenched by NADH + GTP. In contrast the dansyl derivatives of the monocarboxylic amino acids (whose oxidation is activated by the conformational change) had their fluorescence enhanced by NADH + GTP. Despite this specificity there were large numbers of dansyl amino acid binding sites on GDH (ca 35 per oligomer).
Comparison of these responses with those of the MNS-amides showed that in order to detect the structural change in GDH, a probe needed to be negatively charged and to have the negative charge close to the aromatic part of the probe.
In order to detect energy dependent changes in SMP a probe again needed to be negatively charged. However, in this case the negative charge did not have to be as close to the aromatic part of the probe as with the GDH system. Neutral, non-polar probes bind in the lipid phase of the membrane where they cannot sense energy dependent changes in SMP or ionic strength changes in erythrocyte stroma. The negative charge is important in directing the probe to the area of the membrane where it can sense structural changes. In order to test what type of negative charge was required, three 9-anilino acridine derivatives with different acidic groups in the aniline ring were synthesised. Only the sulphonate derivative responded to energy dependent changes in SMP, indicating a preference for this group rather than carboxylate or arsonate.
b) (i) A limited amount of information was obtained concerning GDH. L-Glu enhanced the fluorescence of bound NADH, while D-Glu had no effect and andalpha;Kg was a potent quencher. These sharp differences are difficult to explain since all three molecules bind at the same site on GDH. andalpha;Kg also shifted the conformational equilibrium, making GTP much more effective at inducing the conformational change. The reason for this is as yet unclear.
(ii) The responses of the polarity sensitive probes ANS and MNS, and of the excimer forming probe PS (pyrene-3-sulphonate) to the energy dependent changes in SMP were studied and compared. ANS and MNS bound rapidly to the membrane surface and slowly diffused to internal sites. The energy dependent response was a property of the internal sites only. On binding to SMP no excimer formation by PS was detectable. However, on energisation excimer fluorescence appeared, accompanied by a decrease in monomer fluorescence. This must be due to either an increase in PS concentration within the membrane or to decreased viscosity.
Comparison of the rates of fluorescence decrease following uncoupling revealed a common fast phase (tandfrac12; = 2 sec) with a slow phase characteristic of each probe (tandfrac12; = 12 (ANS), 30 (PS), 60 (MNS) secs). The rates of the slow phases of ANS and PS correlated well with those measured for probe efflux by independent methods. This suggested that the fast phase was a rapid membrane change, followed by a efflux of probe from the membrane (slow phase).
Binding parameters for ANS and MNS were measured by conventional fluorescence techniques. There was an increase in binding and in quantum yield of bound ANS and MNS on energisation of SMP. A novel filtration method was developed for measuring PS binding and showed a two fold increase in PS bound to SMP after energisation. However, this increase was not large enough to account for the observed excimer fluorescence. The technique was also used to verify the fluorescence results for ANS binding.
By measuring fluorescence polarisation and life-times of ANS and MNS in resting and energised SMP, it was possible to rule out viscosity changes as a cause of the PS fluorescence changes. It was therefore proposed that all the probe effects could be explained if energisation involved expulsion of water from the membrane.
This hypothesis was tested by examining the energy dependent responses of ANS and MNS in D2O and H2O. in free solution D2O quenches ANS (and MNS) fluorescence less than H2O giving an isotope effect of 2.8 (2.4). This effect was lowered for both probes on binding to SMP. On energisation, there was a further lowering of the isotope effect on MNS. This suggested that the internal sites at which ANS and MNS bind are partially excluded from water and that on energisation this exclusion increases.
By comparing ANS with NPN (N-phenyl-1-naphthylamine) and other neutral probes, it was shown that the probe binding sites are close to the membrane protein, and are probably at interfaces between hydrophobic and aqueous regions within the membrane, i.e. in an area where the probe could easily sense water exclusion.
A theory of the energised state of SMP was proposed, based on the suggestion that energisation involves water expulsion. The theory accounted for the observed probe responses, as well as for proton uptake, and provided a driving force for phosphorylation.
One probe ABS (2-methoxy,6-chloro,9-acridinyl-p-amino benzene sulphonate) had an electron transport dependent response (as well as an energy dependent one), which appeared to follow the rate of oxidation of coenzyme Q.