An experimental procedure has been devised to record simultaneously fluorescence intensity and fluorescence anisotropy. A photoelastic modulator on the excitation beam enables the anisotropy signal to be recorded in one pass using a single photomultiplier tube and eliminates the need for a polarizer on the emission path. In conjunction with a stopped-flow mixer, providing a time-resolved capability, this procedure was used to study the refolding of apo alpha-lactalbumin following dilution from guanidinium chloride. Although the fluorescence intensity does not change detectably, the fluorescence anisotropy was found to resolve the conformational changes occurring between the initial unfolded state and the molten globule state formed either kinetically during refolding at pH 7.0 or at equilibrium at pH 2.0 (A-state). This result provides further evidence that fluorescence anisotropy is a valuable probe of protein structural transitions and that the information it provides concerning the rotational mobility of a fluorophore can be complementary to the information about the local environment provided by fluorescence intensity.

Fluorescence lifetime is an intrinsic parameter describing the fluorescence process. Changes in the fluorophore's physicochemical environment can lead to changes in the fluorescence lifetime. When used as the readout in biological assays, it is thought to deliver superior results to conventional optical readouts. Hence it has the potential to replace readout technologies currently established in drug discovery such as absorption, luminescence or fluorescence intensity. Here we report the development of an activity assay for human kallikrein 7, a serine protease involved in skin diseases. As a probe, we have selected a blue-fluorescent acridone dye, featuring a remarkably long lifetime that can be quenched by either of the 2 natural amino acids, tyrosine and tryptophan. Incorporating this probe and 1 of the quenching amino acids on either side of the scissile bond of the substrate peptide enables us to monitor the enzymatic activity by quantifying the increase in the fluorescence lifetime signal. A systematic investigation of substrate structures has led to a homogenous, microplate-based, compound profiling assay that yields inhibitory constants down into the single-digit nanomolar range. This type of assay has now been added to our standard portfolio of screening techniques, and is routinely used for compound profiling.

In the absence of bound peptide ligands, major histocompatibility complex (MHC) class I molecules are unstable. In an attempt to determine the minimum requirement for peptide-dependent MHC class I stabilization, we have used short synthetic peptides derived from the Sendai virus nucleoprotein epitope (residues 324 -332, 1 FAPGNYPAL 9 ) to promote its folding in vitro of H-2D b . We found that H-2D b can be stabilized by the pentapeptide 5 NYPAL 9 , which is equivalent to the C-terminal portion of the optimal nonapeptide and includes both the P5 and P9 anchor residues. We have crystallized the complex of the H-2D b molecule with the pentamer and determined the structure to show how a quasi-stable MHC class I molecule can be formed by occupancy of a single binding pocket in the peptide-binding groove. Major histocompatibility complex (MHC)5 class I molecules have evolved to present peptide epitopes of 8 -10 amino acids to cytotoxic T cells. Many MHC class I⅐peptide structures have now been solved by x-ray crystallography, and they all have a common tertiary structure (1). The structure consists of a polymorphic heavy chain (HC) and a nonpolymorphic light chain ␤2-microglobulin (␤2-m), noncovalently associated to form a molecule with two membrane proximal Ig-like domains (the ␣3 and ␤2-m domains) that support a membrane distal ␣1-␣2 "superdomain." The peptide-binding site is formed by a deep cleft between two ␣-helices in this superdomain. Antigenic peptides are always bound in the same orientation, with their N and C termini lying buried deep in pockets that define the ends of the peptide-binding groove (the A and F pockets, respectively). In addition, so-called "anchor residues" make allele specific interactions with polymorphic class I residues located deep inside the binding groove, in "specificitydetermining pockets."The peptide⅐MHC class I complex is formed in the endoplasmic reticulum (ER) and marks the end point of antigen processing (2). During antigen processing, proteins are unfolded and partially hydrolyzed in the cytoplasm, and the resulting polypeptides (of between 8 and 40 amino acids) are translocated across the ER membrane by the transporter associated with antigen processing. Once in the ER, some long peptide epitope precursors can undergo further trimming by the aminopeptidase ERAAP (3, 4) and are selected for assembly with newly synthesized MHC class I molecules that is dependent on their interaction with cofactor molecules such as calreticulin, tapasin, and ERp57. The process results in the preferential release from the ER of class I molecules presenting peptides that bind stably. Recent evidence suggests that this selection of high affinity peptides in vivo may occur by a mechanism that is more complex and controlled than simple competition between potential ligands for binding to class I in the ER (2) and may involve editing of the MHC-bound peptide repertoire in the early secretory pathway of antigen-presenting cells.It is not known whether the loading or editing of class I MHC peptide cargo...

We present a novel homogeneous in vitro assay format and apply it to the quantitative determination of the enzymatic activity of a tyrosine kinase. The assay employs a short peptidic substrate containing a single tyrosine and a single probe attached via a cysteine side chain. The structural flexibility of the peptide allows for the dynamic quenching of the probe by the nonphosphorylated tyrosine side chain. The probe responds with changes in its fluorescence lifetime depending on the phosphorylation state of the tyrosine. We use this effect to directly follow the enzymatic phosphorylation of the substrate, without having to resort to additional assay components such as an antibody against the phosphotyrosine. As an example for the application of this assay principle, we present results from the development of an assay for Abelson kinase (c-Abl) used for compound profiling. Adjustments in the peptide sequence would make this assay format suitable to a wide variety of other tyrosine kinases.