The catalytic behaviour of alpha-CT (alpha-chymotrypsin) is affected by cationic micelles of CTABr (hexadecyltrimethylammonium bromide). The enzyme-micelle interaction leads to an increase in both the V(max) and the affinity for the substrate p -nitrophenyl acetate, indicating higher catalytic efficiency for bound alpha-CT. The bell-shaped profile of alpha-CT activity with increasing CTABr concentrations suggests that the micelle-bound enzyme reacts with the free substrate. Although more active with CTABr micelles, the enzyme stability is essentially the same as observed in buffer only. Enzyme activation is accompanied by changes in alpha-CT conformation. Changes in tertiary structure were observed by the increase in intensity and the red shift in the alpha-CT tryptophan fluorescence spectrum, suggesting the annulment of internal quenching and a more polar location of tryptophan residues. Near-UV CD also indicated the transfer of aromatic residues to a more flexible environment. CTABr micelles also induces an increase in alpha-helix, as seen by far-UV CD and FTIR (Fourier-transform infrared) spectroscopies. The far-UV CD spectrum of alpha-CT shows an increase in the intensity of the positive band at 198 nm and in the negative band at 222 nm, indicating an increased alpha-helical content. This is in agreement with FTIR studies, where an increase in the band at 1655 cm(-1), corresponding to the alpha-helix, was shown by fitting analysis and difference spectroscopy. Spectral deconvolution indicated a reduction in the beta-sheet content in micelle-bound alpha-CT. Our data suggest that the higher catalytic efficiency of micelle-bound alpha-CT results from significant conformational changes.

The flagellar calcium-binding protein (FCaBP) of the flagellated protozoan Trypanosoma cruzi associates with the flagellar membrane via its N-terminal myristate and palmitate moieties in a calcium-modulated, conformation-dependent manner. This mechanism of localization is similar to that described for neuronal calcium sensors, which undergo calcium-dependent changes in conformation, which modulate the availability of the acyl groups for membrane interaction and partner association. To test whether FCaBP undergoes a calcium-dependent conformational change and to explore the role of such a change in flagellar targeting, we first introduced point mutations into each of the two EF-hand calcium-binding sites of FCaBP to define their affinities. Analysis of recombinant EF-3 mutant (E151Q), EF-4 mutant (E188Q), and double mutant proteins showed EF-3 to be the high affinity site (K d ϳ9 M) and EF-4 the low affinity site (K d ϳ120 M). These assignments also correlated with partial (E188Q), nearly complete (E151Q), and complete (E151Q,E188Q) disruption of calcium-induced conformational changes determined by NMR spectrometry. We next expressed the FCaBP E151Q mutant and the double mutant in T. cruzi epimastigotes. These transproteins localized to the flagellum, suggesting the existence of a calcium-dependent interaction of FCaBP that is independent of its intrinsic calcium binding capacity. Several proteins were identified by FCaBP affinity chromatography that interact with FCaBP in a calcium-dependent manner, but with differential dependence on calcium-binding by FCaBP. These findings may have broader implications for the calcium acyl switch mechanism of protein regulation. Flagellar calcium-binding protein (FCaBP)2 is a 24-kDa highly immunogenic protein found in the flagellum of the protozoan parasite Trypanosoma cruzi (1). FCaBP contains four EF-hand calcium-binding domains, the third and fourth (EF-3 and EF-4) of which bind calcium (2). The protein is modified at the N terminus by the addition of myristate and palmitate, both of which are required for association with the inner leaflet of the flagellar membrane (3). Calcium is required for stable flagellar localization as well, since FCaBP can be washed out of detergent-permeabilized trypanosomes if calcium chelators are included in the wash solutions. Because of the size, acylation, calcium binding, membrane association, and calcium-dependent localization of FCaBP, we concluded that the protein is a calcium acyl switch protein. These proteins undergo calcium-dependent membrane association by virtue of calcium-regulated extrusion or sequestration of a myristate moiety that mediates membrane binding (4).The best studied calcium acyl switch protein is recoverin (Rv), a myristoylated calcium-binding protein of the mammalian retina (5) that inhibits rhodopsin kinase (RK) (6) and regulates recovery of the photoreceptor cell after photoexcitation (7). Rv regulates RK via the calcium myristoyl switch mechanism (4). In the resting state of the cell, Rv binds two calcium ions a...

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We introduce a new experimental technique referred to as ellipsometric Raman spectroscopy (ERS) to quantify the Raman optical activity (ROA) where ellipsometry is combined with Raman spectroscopy. The Stokes parameters were determined for Raman scattered light using Fourier decomposition in measurements with achromatic optical components in a nine-point method. We tested the methodology with ultrapure water, a well-known chiral alcohol, and a chiral polymer, and we show that ERS allows for studying not only the vicinities of chiral carbons but also molecular species coupled to form chiral structures. Furthermore, in ERS, isotropic and anisotropic scattering contribute equally, thus making it possible to analyze the low-wavenumber region in samples such as the chiral polymer studied here, for which the ROA signal probably arises mainly from isotropic scattering.