Peptide binding to HLA-A2 and HLA-B27 isolated from Escherichia coli. Reconstitution of HLA-A2 and HLA-B27 heavy chain/beta 2-microglobulin complexes requires specific peptides.

The specificity of peptide binding by human leukocyte antigen (HLA) class I molecules was investigated in a cell-free direct-binding assay. Peptides were assessed for binding to HLA-A2 and HLA-B27 by measuring the formation of heterotrimeric HLA complexes that consisted of iodinated beta 2-microglobulin, HLA heavy chain fragments isolated from the Escherichia coli cytoplasm, and peptide. In this system, no detectable HLA heavy chain-beta 2-microglobulin complexes were formed unless appropriate peptides were intentionally added to the reconstitution solution. Analysis with monoclonal antibodies demonstrated that these heterotrimeric complexes were correctly folded. Five nonhomologous peptides, known to form complexes with HLA-A2 or HLA-B27 from T-cell functional studies, were tested for their capacity to bind to HLA-A2 and HLA-B27 using the reconstitution assay. Four of the peptides bound to the appropriate class I molecule only. One peptide and some (but not all) substitution analogs of it bound to both HLA-A2 and HLA-B27. The effect of peptide length on binding to HLA-B27 was studied, and it was found that the optimal length was 9 or 10 amino acid residues; however, one peptide that bound to HLA-B27 was 15 amino acids long. All peptides that bound to HLA-B27 in the direct-binding assay also competed with antigenic peptides for binding to HLA-B27 on the surface of intact cells, as determined by a standard cytotoxic T-lymphocyte functional assay. Thus, we conclude that HLA-A2 and HLA-B27 bind distinct but partially overlapping sets of peptides and that, at least in vitro, the assembly of HLA heavy chain-beta 2-microglobulin complexes requires specific peptides.     

Cutting edge: HLA-B27 can form a novel beta 2-microglobulin-free heavy chain homodimer structure

HLA-B27 has a striking association with inflammatory arthritis. We show that free HLA-B27 heavy chains can form a disulfide-bonded homodimer, dependent on residue Cys67 in their extracellular alpha 1 domain. Despite the absence of beta 2-microglobulin, HLA-B27 heavy chain homodimers (termed HC-B27) were stabilized by a known peptide epitope. HC-B27 complexes were recognized by the conformation-specific Ab W6/32, but not the ME1 Ab. Surface labeling and immunoprecipitation demonstrated the presence of similar W6/32-reactive free heavy chains at the surface of HLA-B27-transfected T2 cells. HC-B27 homodimer formation might explain the ability of HLA-B27 to induce spondyloarthropathy in beta 2-microglobulin-deficient mice.     

Coexpression of the human HLA-A2 or HLA-B7 heavy chain gene and human beta 2-microglobulin gene in L cells

L cells expressing human HLA-A2 or HLA-B7 class I antigen heavy chains are not recognized by human cytotoxic T lymphocytes directed at HLA-A2 or HLA-B7 antigens. To test whether the absence of human beta 2-m was the cause of the lack of recognition by the human cytotoxic T lymphocytes, coexpression of the human beta 2-m gene and the HLA-A2 or HLA-B7 heavy chain in L cells ("double transfectants") was obtained. In addition, L cells expressing HLA-A2 or HLA-B7 antigens in association with human beta 2-m were obtained by an exchange reaction, in which human beta 2-m from serum replaced the endogenous murine beta 2-m. Both types of transfectant cells were used in 51Cr-release assays and cold target inhibition assays for human cytotoxic T cell clones which were directed at HLA-A2 or HLA-B7. Neither human CTL clones nor a mixture of CTL specific for HLA-A2 and HLA-B7 were able to recognize these cells. Several alternative explanations for these observations are discussed.     

Hierarchical assembly of beta2-microglobulin amyloid in vitro revealed by atomic force microscopy

The kinetics of spontaneous assembly of amyloid fibrils of wild-type beta(2)-microglobulin (beta(2)M) in vitro, under acid conditions (pH 2.5) and low ionic strength, has been followed using thioflavin-T (ThT) binding. In parallel experiments, the morphology of the different fibrillar species present at different time-points during the growth process were characterised using tapping-mode atomic force microscopy (TM-AFM) in air and negative stain electron microscopy (EM). The thioflavin-T assay shows a characteristic lag phase during which the nucleation of fibrils occurs before a rapid growth in fibril density. The volume of fibrils deposited on mica measured from TM-AFM images at each time-point correlates well with the fluorescence data. TM-AFM and negative-stain EM revealed the presence of various kinds of protein aggregates in the lag phase that disappear concomitantly with a rise in the density of amyloid fibrils, suggesting that these aggregates precede fibril growth and may act as nucleation sites. Three distinct morphologies of mature amyloid fibrils were observed within a single growth experiment, as observed previously for the wild-type protein and the variant N17D. Additional supercoiled morphologies of the lower-order fibrils were observed. Comparative height analysis from the TM-AFM data allows each of the mature fibril types and single protofilaments to be identified unambiguously, and reveals that the assembly occurs via a hierarchy of morphological states.     

Recognition of classical and heavy chain forms of HLA-B27 by leukocyte receptors

As an MHC class I protein, the disease association of HLA-B27 with inflammatory arthritis has been widely assumed to imply a role for the T cell antigen receptor (TCR) in disease. However, in addition to their classical antigen-presenting role, HLA class I proteins are recognised by members of the killer immunoglobulin receptor (KIR) and leukocyte immunoglobulin-like receptor (LILR/ILT/LIR) families. Unusual properties of HLA-B27 include an ability of free heavy chains (FHC) to reach the cell surface in the absence of beta2m and to maintain their peptide-binding groove in vitro. This review describes immunomodulatory receptors that recognise HLA class I, and the recognition of HLA-B27 in both the classical beta2m-associated and beta2m-independent forms by members of the KIR and LILR families. Alternative recognition of different forms of HLA-B27 by leukocyte receptors could influence the function of cells from both innate and adaptive immune systems, and may indicate a role for various leukocyte populations in HLA-B27-associated inflammatory disease.     

Induction of HLA-B27 heavy chain homodimer formation after activation in dendritic cells

Introduction

Ankylosing spondylitis (AS) is a severe, chronic inflammatory arthritis, with a strong association to the human major histocompatibilty complex (MHC) class I allele human leucocyte antigen (HLA) B27. Disulfide-linked HLA-B27 heavy-chain homodimers have been implicated as novel structures involved in the aetiology of AS. We have studied the formation of HLA-B27 heavy-chain homodimers in human dendritic cells, which are key antigen-presenting cells and regulators of mammalian immune responses.

Method

Both an in vitro dendritic-like cell line and monocyte-derived dendritic cells from peripheral blood were studied. The KG-1 dendritic-like cell line was transfected with HLA-B27 cDNA constructs, and the cellular distribution, intracellular assembly and ability of HLA-B27 to form heavy-chain homodimers was compared with human monocyte-derived dendritic cells after stimulation with bacterial lipopolysaccharide (LPS).

Results

Immature KG-1 cells expressing HLA-B27 display an intracellular source of MHC class I heavy-chain homodimers partially overlapping with the Golgi bodies, but not the endoplasmic reticulum, which is lost at cell maturation with phorbyl-12-myristate-13-acetate (PMA) and ionomycin. Significantly, the formation of HLA-B27 homodimers in transfected KG-1 cells is induced by maturation, with a transient induction also seen in LPS-stimulated human monocyte-derived dendritic cells expressing HLA-B27. The weak association of wildtype HLA-B*2705 with the transporter associated with antigen processing could also be enhanced by mutation of residues at position 114 and 116 in the peptide-binding groove to those present in the HLA-B*2706 allele.

Conclusion

We have demonstrated that HLA-B27 heavy-chain homodimer formation can be induced by dendritic cell activation, implying that these novel structures may not be displayed to the immune system at all times. Our data suggests that the behaviour of HLA-B27 on dendritic cells may be important in the study of inflammatory arthritis.     

An N-linked glycan modulates the interaction between the CD1d heavy chain and beta 2-microglobulin

Human CD1d molecules consist of a transmembrane CD1 (cluster of differentiation 1) heavy chain in association with beta(2)-microglobulin (beta(2)m). Assembly occurs in the endoplasmic reticulum (ER) and involves the initial glycan-dependent association of the free heavy chain with calreticulin and calnexin and the thiol oxidoreductase ERp57. Folding and disulfide bond formation within the heavy chain occurs prior to beta(2)m binding. There are four N-linked glycans on the CD1d heavy chain, and we mutated them individually to ascertain their importance for the assembly and function of CD1d-beta(2)m heterodimers. None of the four were indispensable for assembly or the ability to bind alpha-galactosyl ceramide and to present it to human NKT cells. Nor were any required for the CD1d molecule to bind and present alpha-galactosyl ceramide after lysosomal processing of a precursor lipid, galactosyl-(alpha1-2)-galactosyl ceramide. However, one glycan, glycan 2 at Asn-42, proved to be of particular importance for the stability of the CD1d-beta(2)m heterodimer. A mutant CD1d heavy chain lacking glycan 2 assembled with beta(2)m and transported from the ER more rapidly than wild-type CD1d and dissociated more readily from beta(2)m upon exposure to detergents. A mutant expressing only glycan 1 dissociated completely from beta(2)m upon exposure to the detergent Triton X-100, whereas a mutant expressing only glycan 2 at Asn-42 was more stable. In addition, glycan 2 was not processed efficiently to the complex form in mature wild-type CD1d molecules. Modeling the glycans on the published structure indicated that glycan 2 interacts significantly with both the CD1d heavy chain and beta(2)m, which may explain these unusual properties.     

A dominant negative mutant beta 2-microglobulin blocks the extracellular folding of a major histocompatibility complex class I heavy chain

The major histocompatibility complex class I (MHC1) molecule plays a crucial role in cytotoxic lymphocyte function. beta 2-Microglobulin (beta 2m) has been demonstrated to be both a structural component of the MHC1 complex and a chaperone-like molecule for MHC1 folding. beta 2m binding to an isolated alpha 3 domain of MHC1 heavy chain at micromolar concentrations has been shown to accurately model the biochemistry and thermodynamics of beta 2m-driven MHC1 folding. These results suggested a model in which the chaperone-like role of beta 2m is dependent on initial binding to the alpha 3 domain interface of MHC1 with beta 2m. Such a model predicts that a mutant beta 2m molecule with an intact MHC1 alpha 3 domain interaction but a defective MHC1 alpha 1 alpha 2 domain interaction would block beta2m-driven folding of MHC1. In this study we generated such a beta 2m mutant and demonstrated that it blocks MHC1 folding by normal beta 2m at the expected micromolar concentrations. Our data support an initial interaction of beta 2m with the MHC1 alpha 3 domain in MHC1 folding. In addition, the dominant negative mutant beta 2m can block T-cell functional responses to antigenic peptide and MHC1.     

HLA-B27 in inbred and non-inbred transgenic mice. Cell surface expression and recognition as an alloantigen in the absence of human beta 2-microglobulin

A gene encoding the H chain of the human class I MHC Ag HLA-B27 was introduced into the germ lines of inbred C57BL/6 (B6) and non-inbred (B6 X SJL/J) F2 mice. By immunofluorescence and flow cytometry, the HLA-B27 gene product was expressed on lymphoid cells at levels comparable to the endogenous H-2b and H-2s class I MHC molecules. In both primary and secondary MLC between responder spleen cells from non-transgenic (B6 X SJL/J) F1 mice and transgenic stimulator cells, CTL were generated that specifically lysed mouse L cell (H-2k) or human B cell targets expressing HLA-B27, and this lysis thus appeared largely unrestricted by H-2. These results indicate that transgenic mice express a functional HLA-B27 gene product on cell surfaces in the absence of the human beta 2-microglobulin gene. These transgenic mice promise to be a valuable resource in the investigation of the unique role of HLA-B27 in inflammatory human disease.     

Independent and synergistic effects of disulfide bond formation, beta 2-microglobulin, and peptides on class I MHC folding and assembly in an in vitro translation system.

We have examined the post-translational processing, intrachain disulfide bond formation, folding, and assembly of MHC class I H chains with beta 2-microglobulin after coupled in vitro translation of homogeneous mRNA and transport of nascent chains into canine microsomal vesicles. The formation of native alpha 3 domain conformation was dependent on conditions that optimized intrachain disulfide bond formation, and efficient folding of the alpha 1 alpha 2 domain required exposure to antigenic peptide. beta 2-microglobulin and peptide acted synergistically in forming native alpha 1 alpha 2 domain structure, and a small proportion of molecules with native alpha 1 alpha 2, but non-native alpha 3 structure were detected, indicating that alpha 3 domain folding is not an absolute prerequisite for the formation of native alpha 1 alpha 2 domain structure.     

PCR/DGGE and 16S rRNA gene library analysis of the colonic microbiota of HLA-B27/beta2-microglobulin transgenic rats

AIMS: To determine the phylogenetic composition of the colonic microbiota of transgenic (TG) HLA-B27 rats using 16S ribosomal RNA (rRNA) gene sequences obtained from denaturing gradient gel electrophoresis (DGGE) gels and sequences from a 16S rRNA gene library. METHODS AND RESULTS: Colonic microbiota of TG and nontransgenic (NT) rats harboured by 10-week-old and 6-month-old animals was screened using PCR/DGGE. Six months old TG rats had marked inflammation of the colon compared with 10-week-old TG and NT rats. The DGGE profiles of rats with inflamed colon were similar from rat to rat (Dice's Similarity Coefficient proximal colon 73%, distal colon 83%) whereas profiles from animals without inflammation were dissimilar (52-64%). Identifications of bacterial origins of 16S rRNA gene sequences obtained from DGGE gels (200 bp) and from 16S rRNA clones (450 bp) of the colonic microbiota of diseased rats gave sequences most closely phylogenetically affiliated with uncultured or unknown bacteria. CONCLUSIONS: PCR/DGGE was shown to be an effective method to compare the colonic microbiota composition of TG and NT rats relative to the progression of inflammatory disease. Sequencing of 16S rRNA gene fragments from DGGE gels or 16S rRNA gene clones from a random library showed that uncultured or unknown bacteria were most commonly detected by both methods. It can be concluded that it would be better in future studies to search for the antigens produced by the gut microbiota against which the dysfunctional immune system reacts rather than seek phylogenetic associations. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR/DGGE can be used as a rapid initial screening method to compare the composition of bacterial communities of initially unknown composition that are associated with the development of intestinal disease.     

Inhibition of heavy chain and beta2-microglobulin synthesis as a mechanism of major histocompatibility complex class I downregulation during Epstein-Barr virus replication

The mechanisms of major histocompatibility complex (MHC) class I downregulation during Epstein-Barr virus (EBV) replication are not well characterized. Here we show that in several cell lines infected with a recombinant EBV strain encoding green fluorescent protein (GFP), the virus lytic cycle coincides with GFP expression, which thus can be used as a marker of virus replication. EBV replication resulted in downregulation of MHC class II and all classical MHC class I alleles independently of viral DNA synthesis or late gene expression. Although assembled MHC class I complexes, the total pool of heavy chains, and beta2-microglobulin (beta2m) were significantly downregulated, free class I heavy chains were stabilized at the surface of cells replicating EBV. Calnexin expression was increased in GFP+ cells, and calnexin and calreticulin accumulated at the cell surface that could contribute to the stabilization of class I heavy chains. Decreased expression levels of another chaperone, ERp57, and TAP2, a transporter associated with antigen processing and presentation, correlated with delayed kinetics of MHC class I maturation. Levels of both class I heavy chain and beta2m mRNA were reduced, and metabolic labeling experiments demonstrated a very low rate of class I heavy chain synthesis in lytically infected cells. MHC class I and MHC class II downregulation was mimicked by pharmacological inhibition of protein synthesis in latently infected cells. Our data suggest that although several mechanisms may contribute to MHC class I downregulation in the course of EBV replication, inhibition of MHC class I synthesis plays the primary role in the process.     

Tapasin enhances assembly of transporters associated with antigen processing-dependent and -independent peptides with HLA-A2 and HLA-B27 expressed in insect cells.

Assembly of HLA class I-peptide complexes is assisted by multiple proteins that associate with HLA molecules in loading complexes. These include the housekeeping chaperones calnexin and calreticulin and two essential proteins, the transporters associated with antigen processing (TAP) for peptide supply, and the protein tapasin which is thought to act as a specialized chaperone. We dissected functional effects of processing cofactors by co-expressing in insect cells various combinations of the human proteins HLA-A2, HLA-B27, beta(2)-microglobulin, TAP, calnexin, calreticulin, and tapasin. Stability at 37 degrees C and surface expression of class I dimers correlated closely in baculovirus-infected Sf9 cells, suggesting that these cells retain empty dimers in the endoplasmic reticulum. Both HLA molecules form substantial quantities of stable complexes with insect cell-produced peptide pools. These pools are TAP-selected cytosolic peptides for HLA-B27 but endoplasmic reticulum-derived, i.e. TAP-independent peptides for HLA-A2. This discrepancy may be due to peptide selection by human TAP which is much better adapted to the HLA-B27 than to the HLA-A2 ligand preferences. HLA class I assembly with peptides from TAP-dependent and -independent pools was enhanced strongly by tapasin. Thus, tapasin acts as a chaperone and/or peptide editor that facilitates assembly of peptides with HLA class I molecules independently of mediating their interaction with TAP and/or retention in the endoplasmic reticulum.     

Crosses of two independently derived transgenic mice demonstrate functional complementation of the genes encoding heavy (HLA-B27) and light (beta 2-microglobulin) chains of HLA class I antigens

In man a number of diseases are associated with certain alleles of MHC antigens. The most pronounced example is ankylosing spondylitis, which is strongly associated with HLA-B27. As a first step towards a model system to study the basis of this association, transgenic mice were generated that showed cell surface expression of the HLA-B27 antigen biochemically indistinguishable from HLA-B27 antigen expressed on human cells. This result was obtained by crossing two independently derived strains of mice, one of which is transgenic for the HLA-B27 heavy chain gene, and the other carrying and expressing the human beta 2m gene. Examination of HLA-B27 and human beta 2m mRNA in various tissues shows the two genes to be expressed in a coordinate fashion. The mRNA levels follow those of endogenous H-2 Class I genes.     

Spontaneous inflammatory disease in transgenic rats expressing HLA-B27 and human beta 2m: an animal model of HLA-B27-associated human disorders

Humans who have inherited the human class I major histocompatibility allele HLA-B27 have a markedly increased risk of developing the multi-organ system diseases termed spondyloarthropathies. To investigate the role of B27 in these disorders, we introduced the B27 and human beta 2-microglobulin genes into rats, a species known to be quite susceptible to experimentally induced inflammatory disease. Rats from one transgenic line spontaneously developed inflammatory disease involving the gastrointestinal tract, peripheral and vertebral joints, male genital tract, skin, nails, and heart. This pattern of organ system involvement showed a striking resemblance to the B27-associated human disorders. These results establish that B27 plays a central role in the pathogenesis of the multi-organ system processes of the spondyloarthropathies. Elucidation of the role of B27 should be facilitated by this transgenic model.     

Translocation of branched-chain arginine peptides through cell membranes: flexibility in the spatial disposition of positive charges in membrane-permeable peptides

A basic peptide derived from HIV-1 Tat has been reported to have the ability to translocate through cell membranes and to bring exogenous proteins into cells. We have demonstrated that these features could be observed among many arginine-rich peptides, and the presence of a ubiquitous internalization mechanism for arginine-rich oligopeptides has been suggested. In this report, we report that these features are also applicable to the peptides having branched-chain structures. Peptides that have arginine residues on four branched chains (R(n))(4) [n (number of arginine residues)= 0-6] were prepared. Fluorescence microscopic observation revealed that the (R(2))(4) peptide exhibited the most efficient translocation. The dependence on the number of arginine residues of the translocation efficiency and cellular localization was also observed for the branched-chain peptides as was seen in the linear peptides. Quite interestingly, efficient translocation was also recognized in the (RG(3)R)(4) peptide, where three glycine residues intervened between two arginine residues on each chain of (R(2))(4). The results strongly suggested that a linear structure was not indispensable for the translocation of arginine-rich peptides and that there could be considerable flexibility in the location of the arginine residue in the molecules.     

Kinetics and thermodynamics of beta 2-microglobulin binding to the alpha 3 domain of major histocompatibility complex class I heavy chain

The major histocompatibility complex (MHC) class I molecule plays a crucial role in cytotoxic lymphocyte function. Functional class I MHC exists as a heterotrimer consisting of the MHC class I heavy chain, an antigenic peptide fragment, and beta2-microglobulin (beta2m). beta2m has been previously shown to play an important role in the folding of the MHC heavy chain without continued beta2m association with the MHC complex. Therefore, beta2m is both a structural component of the MHC complex and a chaperone-like molecule for MHC folding. In this study we provide data supporting a model in which the chaperone-like role of beta2m is dependent on initial binding to only one of the two beta2m interfaces with class 1 heavy chain. beta2-Microglobulin binding to an isolated alpha3 domain of the class I MHC heavy chain accurately models the biochemistry and thermodynamics of beta2m-driven refolding. Our results explain a 1000-fold discrepancy between beta2m binding and refolding of MHC1. The biochemical study of the individual domains of complex molecules is an important strategy for understanding their dynamic structure and multiple functions.     

HLA-B27 in transgenic rats forms disulfide-linked heavy chain oligomers and multimers that bind to the chaperone BiP

To test the hypothesis that HLA-B27 predisposes to disease by forming disulfide-linked homodimers, we examined rats transgenic for HLA-B27, mutant Cys(67)Ser HLA-B27, or HLA-B7. In splenic Con A blasts from high transgene copy B27 lines that develop inflammatory disease, the anti-H chain mAb HC10 precipitated four bands of molecular mass 78-105 kDa and additional higher molecular mass material, seen by nonreducing SDS-PAGE. Upon reduction, all except one 78-kDa band resolved to 44 kDa, the size of the H chain monomer. The 78-kDa band was found to be BiP/Grp78, and the other high molecular mass material was identified as B27 H chain. Analysis of a disease-resistant low copy B27 line showed qualitatively similar high molecular mass bands that were less abundant relative to H chain monomer. Disease-prone rats with a Cys(67)Ser B27 mutant showed B27 H chain bands at 95 and 115 kDa and a BiP band at 78 kDa, whereas only scant high molecular mass bands were found in cells from control HLA-B7 rats. (125)I-surface labeled B27 oligomers were immunoprecipitated with HC10, but not with a mAb to folded B27-beta(2)-microglobulin-peptide complexes. Immunoprecipitation of BiP with anti-BiP Abs coprecipitated B27 H chain multimers. Folding and maturation of B27 were slow compared with B7. These data indicate that disulfide-linked intracellular H chain complexes are more prone to form and bind BiP in disease-prone wild-type B27 and B27-C67S rats than in disease-resistant HLA-B7 rats. The data support the hypothesis that accumulation of misfolded B27 participates in the pathogenesis of B27-associated disease.     

Secondary structure of the murine histocompatibility alloantigen H-2Kb: relationship between heavy chain, beta 2-microglobulin, and antigenic reactivity

The far-ultraviolet circular dichroism (CD) spectra of the extracellular portion (papain-cleaved fragment) of the histocompatibility antigen H-2Kb and its noncovalently associated components, heavy chain and beta 2-microglobulin (beta 2m), indicate that the antigen is highly structured, containing about 30% alpha-helix, 41% beta-sheet, and 29% random coil. Separation of beta 2m from the heavy chain produced a decrease in heavy chain alpha-helix and beta-sheet structure which correlated with a loss of alloantigenic reactivity. Reconstitution of the heavy chain-beta 2m complex resulted in an increase in secondary structure which was greater than the sum of the free chains and the recovery of considerable alloantigenic reactivity. This suggests that some of the secondary structure and much of the alloantigenic reactivity may depend on conformation associated with the binding of beta 2m to heavy chain. A prediction of heavy chain secondary structure based on Chou-Fasman analysis of the primary amino acid sequence agreed with results from CD measurements and suggested that the segments of alpha-helix and beta-sheet structure are distributed throughout the molecule.     

Photophysical analysis of class I major histocompatibility complex protein assembly using a xanthene-derivatized beta2-microglobulin

Spectral changes and a sixfold increase in the emission intensity were observed in the fluorescence of a single xanthene probe (Texas red) attached to beta2m-microglobulin (beta2m) upon assembly of beta2m into a ternary complex with mouse H-2Kd heavy chain and influenza nuclear protein peptide. Dissociation of the labeled beta2m from the ternary complex restored the probe's fluorescence and absorption spectra and reduced the emission intensity. Thus changes in xanthene probe fluorescence upon association/dissociation of the labeled beta2m molecule with/from the ternary complex provide a simple and convenient method for studying the assembly/dissociation mechanism of the class I major histocompatibility complex (MHC-I) encoded molecule. The photophysical changes in the probe can be accounted for by the oligomerization of free labeled beta2m molecules. The fluorescence at 610 nm is due to beta2m dimers, where the probes are significantly separated spatially so that their emission and excitation properties are close to those of xanthene monomers. Fluorescence around 630 nm is due to beta2m oligomers where xanthene probes interact. Minima in the steady-state excitation (550 nm) and emission (630 nm) anisotropy spectra correlate with the maxima of the high-order oligomer excitation and emission spectra, showing that their fluorescence is more depolarized. These photophysical features are explained by splitting of the first singlet excited state of interacting xanthene probes that can be modeled by exciton theory.