SH2db, an information system for the SH2 domain.

SH2 domains are key mediators of phosphotyrosine-based signalling, and therapeutic targets for diverse, mostly oncological, disease indications. They have a highly conserved structure with a central beta sheet that divides the binding surface of the protein into two main pockets, responsible for phosphotyrosine binding (pY pocket) and substrate specificity (pY + 3 pocket). In recent years, structural databases have proven to be invaluable resources for the drug discovery community, as they contain highly relevant and up-to-date information on important protein classes. Here, we present SH2db, a comprehensive structural database and webserver for SH2 domain structures. To organize these protein structures efficiently, we introduce (i) a generic residue numbering scheme to enhance the comparability of different SH2 domains, (ii) a structure-based multiple sequence alignment of all 120 human wild-type SH2 domain sequences and their PDB and AlphaFold structures. The aligned sequences and structures can be searched, browsed and downloaded from the online interface of SH2db (http://sh2db.ttk.hu), with functions to conveniently prepare multiple structures into a Pymol session, and to export simple charts on the contents of the database. Our hope is that SH2db can assist researchers in their day-to-day work by becoming a one-stop shop for SH2 domain related research.

Histone deacetylase inhibitors as a potential new treatment for psoriatic disease and other inflammatory conditions.

Collectively known as psoriatic disease, psoriasis and psoriatic arthritis (PsA) are immune-mediated inflammatory diseases in which patients present with cutaneous and musculoskeletal inflammation. Affecting roughly 2-3% of the world's total population, there remains unmet therapeutic needs in both psoriasis and PsA despite the availability of current immunomodulatory treatments. As a result, patients with psoriatic disease often experience reduced quality of life. Recently, a class of small molecules, commonly investigated as anti-cancer agents, called histone deacetylase (HDAC) inhibitors, have been proposed as a new promising anti-inflammatory treatment for immune- and inflammatory-related diseases. In inflammatory diseases, current evidence is derived from studies on diseases like rheumatoid arthritis (RA) and systematic lupus erythematosus (SLE), and while there are some reports studying psoriasis, data on PsA patients are not yet available. In this review, we provide a brief overview of psoriatic disease, psoriasis, and PsA, as well as HDACs, and discuss the rationale behind the potential use of HDAC inhibitors in the management of persistent inflammation to suggest its possible use in psoriatic disease.

Strategies for over-expression and purification of recombinant full length STAT5B in Escherichia coli.

STAT5B, a ubiquitious transcription factor, has been implicated in the onset and progression of several cancers. Since the inhibition of STAT activity holds significant therapeutic potential, there is a need to develop high-throughput biophysical screening platforms to rapidly identify high affinity binders of STATs. Biophysical assays would benefit from the efficient and cost-effective production of high purity, full-length STAT proteins. Herein, we have sampled a large region of protein expression and purification space that has substantially increased recombinant STAT5B protein yields from Escherichia coli. The identity of STAT5B was confirmed by Western blotting analysis, while the results of a fluorescence polarization assay indicated that the purified protein is correctly folded and functional. A thermal shift assay was employed to assess the effect of various osmolytes on the stability of the protein. The protein expression conditions identified in this study allowed for more efficient and higher recovery of soluble STAT5B protein, which will enable a broad range of biophysical studies and facilitate high-throughput STAT5B drug screening.

ProxyPhos sensors for the detection of negatively charged membranes.

Membrane-embedded negatively charged phospholipids (MENCP) can be used as biomarkers for a range of biological processes, including early detection of apoptosis in animal cells, drug-induced phospholipidosis, and selective detection of bacterial over animal cells. Currently, several technologies for the detection of apoptosis and bacterial cells are based on the recognition of MENCPs, including the AnnexinV stain and PSVue™ probes. As probes, these technologies have limitations, the most significant of which is the need for washing the unbound probe away to achieve optimal signal. In contrast, a turn-on chemosensor selective for MENCP would address this shortcoming, and allow for a more rapid protocol for the detection of apoptosis, bacteria and for other relevant applications. In this work, the aim was to explore whether ProxyPhos chemosensors, previously reported by our group for the detection of proximally phosphorylated peptides and proteins, could be re-purposed for the detection of MENCPs. Six lead ProxyPhos sensors were screened against synthetic vesicles containing biologically relevant negatively charged phospholipids including phosphatidic acid (PA), phosphatidylglycerol (PG), cardiolipin (CL) and phosphatidylserine (PS). Through these screens, ProxyPhos sensors exhibiting high selectivity for the detection of MENCPs over zwitterionic lipids were identified. Particular selectivity was observed for PA and CL. Sensitivity of the lead sensors for MENCPs was suitable for the detection of apoptosis: ProxyPhos detected vesicles containing as little as 2.5% PS and detected camptothecin-induced apoptosis in mammalian cells in flow cytometry experiments. The results suggest that ProxyPhos sensors can be used for the detection of MENCPs in synthetic vesicles and live mammalian cells.

Phosphorylation-dependent changes in nucleotide binding, conformation, and dynamics of the first nucleotide binding domain (NBD1) of the sulfonylurea receptor 2B (SUR2B).

The sulfonylurea receptor 2B (SUR2B) forms the regulatory subunit of ATP-sensitive potassium (KATP) channels in vascular smooth muscle. Phosphorylation of the SUR2B nucleotide binding domains (NBD1 and NBD2) by protein kinase A results in increased channel open probability. Here, we investigate the effects of phosphorylation on the structure and nucleotide binding properties of NBD1. Phosphorylation sites in SUR2B NBD1 are located in an N-terminal tail that is disordered. Nuclear magnetic resonance (NMR) data indicate that phosphorylation of the N-terminal tail affects multiple residues in NBD1, including residues in the NBD2-binding site, and results in altered conformation and dynamics of NBD1. NMR spectra of NBD1 lacking the N-terminal tail, NBD1-ΔN, suggest that phosphorylation disrupts interactions of the N-terminal tail with the core of NBD1, a model supported by dynamic light scattering. Increased nucleotide binding of phosphorylated NBD1 and NBD1-ΔN, compared with non-phosphorylated NBD1, suggests that by disrupting the interaction of the NBD core with the N-terminal tail, phosphorylation also exposes the MgATP-binding site on NBD1. These data provide insights into the molecular basis by which phosphorylation of SUR2B NBD1 activates KATP channels.

Successful development and use of a thermodynamic stability screen for optimizing the yield of nucleotide binding domains.

ATP sensitive potassium (KATP) channels consist of four copies of a pore-forming inward rectifying potassium channel (Kir6.1 or Kir6.2) and four copies of a sulfonylurea receptor (SUR1, SUR2A, or SUR2B). SUR proteins are members of the ATP-binding cassette superfamily of proteins. Binding of ATP to the Kir6.x subunit mediates channel inhibition, whereas MgATP binding and hydrolysis at the SUR NBDs results in channel opening. Mutations in SUR1 and SUR2A NBDs cause diseases of insulin secretion and cardiac disorders, respectively, underlying the importance of studying the NBDs. Although purification of SUR2A NBD1 in a soluble form is possible, the lack of long-term sample stability of the protein in a concentrated form has precluded detailed studies of the protein aimed at gaining a molecular-level understanding of how SUR mutations cause disease. Here we use a convenient and cost-effective thermodynamic screening method to probe stabilizing conditions for SUR2A NBD1. Results from the screen are used to alter the purification protocol to allow for significantly increased yields of the purified protein. In addition, the screen provides strategies for long-term storage of NBD1 and generating NBD1 samples at high concentrations suitable for NMR studies. NMR spectra of NBD1 with MgAMP-PNP are of higher quality compared to using MgATP, indicating that MgAMP-PNP be used as the ligand in future NMR studies. The screen presented here can be expanded to using different additives and can be employed to enhance purification yields, sample life times, and storage of other low stability nucleotide binding domains, such as GTPases.

Inhibitor Library Screening of SH2 Domains Through Denaturation-Based Assays.

Screening of inhibitor libraries for candidate ligands is an important step in the drug discovery process. Thermal denaturation-based screening strategies are built on the premise that a protein-ligand complex has an altered stability profile compared to the protein alone. As such, these assays provide an accessible and rapid methodology for stratifying ligands that directly engage with the protein target of interest. Here, we describe three denaturation-based strategies for examining protein-inhibitor binding, in the context of SH2 domains. This includes conventional dye-based Thermal Shift Assays (TSA), nonconventional labeled ligand-based TSA, and Cellular Thermal Shift Assays (CETSA). Conventional dye-based TSA reports on the fluorescence of an external hydrophobic dye as it interacts with heat-exposed nonpolar protein surfaces as the temperature is incrementally increased. By contrast, nonconventional-labeled ligand TSA involves a fluorescence-tagged probe (phosphopeptide for SH2 domains) that is quenched as it dissociates from the protein during the denaturation process. CETSA involves monitoring the presence of the protein via Western blotting as the temperature is increased. In all three approaches, performing the assay in the presence of a candidate ligand can alter the melting profile of the protein. These assays offer primary screening tools to examine SH2 domain inhibitors libraries with varying chemical motifs, and a subset of the advantages and limitations of each approach is also discussed.

Evaluation of Small-Molecule HDAC Inhibitors Through In Vitro and In Cellulo Approaches.

The aberrant activity of histone deacetylases (HDACs) across a broad range of cancers and other disease indications has led to the development of small-molecule inhibitors that target one or more members of the HDAC protein family. Emerging HDAC inhibitors that show promise in drug discovery programs must be assessed across a range of in vitro assays to establish an inhibitor profile for potency and cellular selectivity towards target HDAC(s) as well as preliminary absorption, distribution, metabolism, and excretion (ADME) features. Here we provide an overview of methods to determine a subset of pivotal in vitro drug-like parameters for HDAC inhibitors (HDACi). We initially describe protocols for parallel artificial membrane permeability assays (PAMPA) to evaluate the passive permeability of small molecules across lipid membranes. Subsequently, we elaborate on cytotoxicity assays using CellTiter-Blue to determine HDACi-induced cell death in healthy/diseased cellular models. We next focus on assessing the target engagement of inhibitors with the appropriate HDAC isoforms in a cellular environment via Western blotting of acetylated HDAC substrates. Finally, we provide detailed guidelines on how to assess the metabolic stability of HDACi through whole blood stability assays. Collectively, these assays provide an overview of the permeability, selectivity, and stability of the HDAC inhibitor under development.

Fluorescence Polarization-Based Competition Assays to Evaluate Histone Deacetylase 6 Inhibitors.

Histone deacetylase 6 (HDAC6) is an emerging clinical target for the treatment of several hematological cancers and central nervous system disorders. HDAC6 catalyzes the deacetylation of lysine residues on substrates such as tubulin, with profound implications in key cellular processes, including cellular motility and migration. This critical deacetylation activity occurs at the catalytic domain 2 (CD2) of HDAC6, and small molecule inhibitors of HDAC6 are designed to target CD2. We briefly highlight previously reported strategies for recombinant bacterial expression and purification of the HDAC6 CD2. We aim to discuss competition assays that have been used to evaluate the potency of potential HDAC6 inhibitors against CD2 via displacement of pre-bound fluorescent HDAC-probes. Moreover, we elaborate on previous protocols that have been employed in inhibitor screening and present an HDAC6-selective probe that also enables rapid and reliable high-throughput screening of new chemical entities designed to target the HDAC6 CD2.

Discovery of YSR734: A Covalent HDAC Inhibitor with Cellular Activity in Acute Myeloid Leukemia and Duchenne Muscular Dystrophy.

Histone deacetylases (HDACs) have emerged as powerful epigenetic modifiers of histone/non-histone proteins via catalyzing the deacetylation of ε-N-acetyl lysines. The dysregulated activity of these Zn2+-dependent hydrolases has been broadly implicated in disease, notably cancer. Clinically, the recurring dose-limiting toxicities of first-generation HDACi sparked a paradigm shift toward safer isoform-specific molecules. With pervasive roles in aggressive diseases, there remains a need for novel approaches to target these enzymes. Herein, we report the discovery of YSR734, a first-in-class covalent HDACi, with a 2-aminobenzanilide Zn2+ chelate and a pentafluorobenzenesulfonamide electrophile. This class I selective proof of concept modified HDAC2Cys274 (catalytic domain), with nM potency against HDAC1-3, sub-µM activity in MV4-11 cells, and limited cytotoxicity in MRC-9 fibroblasts. In C2C12 myoblasts, YSR734 activated muscle-specific biomarkers myogenin/Cav3, causing potent differentiation into myotubes (applications in Duchenne Muscular Dystrophy). Current efforts are focused on improving in vivo ADME toward a preclinical covalent HDACi.

Medicinal chemistry advances in targeting class I histone deacetylases.

Histone deacetylases (HDACs) are a class of zinc (Zn)-dependent metalloenzymes that are responsible for epigenetic modifications. HDACs are largely associated with histone proteins that regulate gene expression at the DNA level. This tight regulation is controlled by acetylation [via histone acetyl transferases (HATs)] and deacetylation (via HDACs) of histone and non-histone proteins that alter the coiling state of DNA, thus impacting gene expression as a downstream effect. For the last two decades, HDACs have been studied extensively and indicated in a range of diseases where HDAC dysregulation has been strongly correlated with disease emergence and progression-most prominently, cancer, neurodegenerative diseases, HIV, and inflammatory diseases. The involvement of HDACs as regulators in these biochemical pathways established them as an attractive therapeutic target. This review summarizes the drug development efforts exerted to create HDAC inhibitors (HDACis), specifically class I HDACs, with a focus on the medicinal chemistry, structural design, and pharmacology aspects of these inhibitors.

NMR and fluorescence studies of drug binding to the first nucleotide binding domain of SUR2A.

ATP sensitive potassium (K(ATP)) channels are composed of four copies of a pore-forming inward rectifying potassium channel (Kir6.1 or Kir6.2) and four copies of a sulfonylurea receptor (SUR1, SUR2A, or SUR2B) that surround the pore. SUR proteins are members of the ATP-binding cassette (ABC) superfamily of proteins. Binding of MgATP at the SUR nucleotide binding domains (NBDs) results in NBD dimerization, and hydrolysis of MgATP at the NBDs leads to channel opening. The SUR proteins also mediate interactions with K(ATP) channel openers (KCOs) that activate the channel, with KCO binding and/or activation involving residues in the transmembrane helices and cytoplasmic loops of the SUR proteins. Because the cytoplasmic loops make extensive interactions with the NBDs, we hypothesized that the NBDs may also be involved in KCO binding. Here, we report nuclear magnetic resonance (NMR) spectroscopy studies that demonstrate a specific interaction of the KCO pinacidil with the first nucleotide binding domain (NBD1) from SUR2A, the regulatory SUR protein in cardiac K(ATP) channels. Intrinsic tryptophan fluorescence titrations also demonstrate binding of pinacidil to SUR2A NBD1, and fluorescent nucleotide binding studies show that pinacidil binding increases the affinity of SUR2A NBD1 for ATP. In contrast, the KCO diazoxide does not interact with SUR2A NBD1 under the same conditions. NMR relaxation experiments and size exclusion chromatography indicate that SUR2A NBD1 is monomeric under the conditions used in drug binding studies. These studies identify additional binding sites for commonly used KCOs and provide a foundation for testing binding of drugs to the SUR NBDs.

Emerging mechanisms of targeted protein degradation by molecular glues.

Targeted protein degradation has emerged as a transformative therapeutic modality for the treatment of human diseases. The clinical successes of approved protein degraders like lenalidomide and thalidomide in cancers and immune disorders, combined with the recent clinical debut of investigational heterobifunctional degraders, have demonstrated the potential of this pharmacological approach to expand the druggable proteome and improve patient outcomes. Molecular glue degraders are a class of protein degraders that operate by recruiting target proteins to cellular degradation machinery via noncanonical protein-protein interactions, inducing the destruction of the target protein. While heterobifunctional degraders consist of two distinct protein-binding moieties connected by a linker, molecular glue degraders contain a single pharmacophore and are thus more synthetically accessible, ligand-efficient, and often possess more drug-like physicochemical properties. In this chapter, we will explore the history of the field-from its conception to the recently accelerating discovery of novel glue degrader mechanisms-and contemplate its trajectory towards rational design with the emergence of new methods for protein quantification and high-throughput assays to screen for novel degraders.

Structural and mutational analysis of member-specific STAT functions.

BACKGROUND: The STAT family of transcription factors control gene expression in response to signals from various stimulus. They display functions in diseases ranging from autoimmunity and chronic inflammatory disease to cancer and infectious disease. SCOPE OF REVIEW: This work uses an approach informed by structural data to explore how domain-specific structural variations, post-translational modifications, and the cancer genome mutational landscape dictate STAT member-specific activities. MAJOR CONCLUSIONS: We illustrated the structure-function relationship of STAT proteins and highlighted their effect on member-specific activity. We correlated disease-linked STAT mutations to the structure and cancer genome mutational landscape and proposed rational drug targeting approaches of oncogenic STAT pathway addiction. GENERAL SIGNIFICANCE: Hyper-activated STATs and their variants are associated with multiple diseases and are considered high value oncology targets. A full understanding of the molecular basis of member-specific STAT-mediated signaling and the strategies to selectively target them requires examination of the difference in their structures and sequences.

Phenotypic Screening of Histone Deacetylase (HDAC) Inhibitors against Schistosoma mansoni.

Schistosomiasis is a prevalent yet neglected tropical parasitic disease caused by the Schistosoma genus of blood flukes. Praziquantel is the only currently available treatment, hence drug resistance poses a major threat. Recently, histone deacetylase 8 (HDAC8) selective inhibitors have been proposed as a viable treatment for schistosomiasis. Herein, we report the phenotypic screening of a focused library of small molecules of varying HDAC isozyme-inhibition profiles, including eight HDAC8 inhibitors with >10-fold selectivity in comparable functional inhibition assays and IC50 values against HDAC8<100 nM. HDAC8-selective inhibitors showed the lowest potency against Schistosoma mansoni newly transformed schistosomula (NTS). Pan-HDAC inhibitors MMH258, MMH259, and MMH373, as assessed by functional inhibition assays, with minimal or no-observed hHDAC8 and SmHDAC8 activities, were active against both NTS (MMH258, IC50 =1.5 µM; MMH259, IC50 =2.3 µM) and adult S. mansoni (MMH258, IC50 =2.1 µM; MMH373, IC50 =3.4 µM). Our results indicate that neither hHDAC8 nor SmHDAC8 activity were directly correlated to their NTS and adult S. mansoni activities.

High Efficacy and Drug Synergy of HDAC6-Selective Inhibitor NN-429 in Natural Killer (NK)/T-Cell Lymphoma.

NK/T-cell lymphoma (NKTCL) and γδ T-cell non-Hodgkin lymphomas (γδ T-NHL) are highly aggressive lymphomas that lack rationally designed therapies and rely on repurposed chemotherapeutics from other hematological cancers. Histone deacetylases (HDACs) have been targeted in a range of malignancies, including T-cell lymphomas. This study represents exploratory findings of HDAC6 inhibition in NKTCL and γδ T-NHL through a second-generation inhibitor NN-429. With nanomolar in vitro HDAC6 potency and high in vitro and in cellulo selectivity for HDAC6, NN-429 also exhibited long residence time and improved pharmacokinetic properties in contrast to older generation inhibitors. Following unique selective cytotoxicity towards γδ T-NHL and NKTCL, NN-429 demonstrated a synergistic relationship with the clinical agent etoposide and potential synergies with doxorubicin, cytarabine, and SNS-032 in these disease models, opening an avenue for combination treatment strategies.

Discovery of HDAC6-Selective Inhibitor NN-390 with in Vitro Efficacy in Group 3 Medulloblastoma.

Histone deacetylase 6 (HDAC6) has been targeted in clinical studies for anticancer effects due to its role in oncogenic transformation and metastasis. Through a second-generation structure-activity relationship (SAR) study, the design, and biological evaluation of the selective HDAC6 inhibitor NN-390 is reported. With nanomolar HDAC6 potency, >200-550-fold selectivity for HDAC6 in analogous HDAC isoform functional assays, potent intracellular target engagement, and robust cellular efficacy in cancer cell lines, NN-390 is the first HDAC6-selective inhibitor to show therapeutic potential in metastatic Group 3 medulloblastoma (MB), an aggressive pediatric brain tumor often associated with leptomeningeal metastases and therapy resistance. MB stem cells contribute to these patients' poor clinical outcomes. NN-390 selectively targets this cell population with a 44.3-fold therapeutic margin between patient-derived Group 3 MB cells in comparison to healthy neural stem cells. NN-390 demonstrated a 45-fold increased potency over HDAC6-selective clinical candidate citarinostat. In summary, HDAC6-selective molecules demonstrated in vitro therapeutic potential against Group 3 MB.

Blocking STAT3/5 through direct or upstream kinase targeting in leukemic cutaneous T-cell lymphoma.

Leukemic cutaneous T-cell lymphomas (L-CTCL) are lymphoproliferative disorders of skin-homing mature T-cells causing severe symptoms and high mortality through chronic inflammation, tissue destruction, and serious infections. Despite numerous genomic sequencing efforts, recurrent driver mutations have not been identified, but chromosomal losses and gains are frequent and dominant. We integrated genomic landscape analyses with innovative pharmacologic interference studies to identify key vulnerable nodes in L-CTCL. We detected copy number gains of loci containing the STAT3/5 oncogenes in 74% (n = 17/23) of L-CTCL, which correlated with the increased clonal T-cell count in the blood. Dual inhibition of STAT3/5 using small-molecule degraders and multi-kinase blockers abolished L-CTCL cell growth in vitro and ex vivo, whereby PAK kinase inhibition was specifically selective for L-CTCL patient cells carrying STAT3/5 gains. Importantly, the PAK inhibitor FRAx597 demonstrated encouraging anti-leukemic activity in vivo by inhibiting tumor growth and disease dissemination in intradermally xenografted mice. We conclude that STAT3/5 and PAK kinase interaction represents a new therapeutic node to be further explored in L-CTCL.

The first nucleotide binding domain of the sulfonylurea receptor 2A contains regulatory elements and is folded and functions as an independent module.

The sulfonylurea receptor 2A (SUR2A) is an ATP-binding cassette (ABC) protein that forms the regulatory subunit of ATP-sensitive potassium (K(ATP)) channels in the heart. ATP binding and hydrolysis at the SUR2A nucleotide binding domains (NBDs) control gating of K(ATP) channels, and mutations in the NBDs that affect ATP hydrolysis and cellular trafficking cause cardiovascular disorders. To date, there is limited information on the SUR2A NBDs and the effects of disease-causing mutations on their structure and interactions. Structural and biophysical studies of NBDs, especially from eukaryotic ABC proteins like SUR2A, have been hindered by low solubility of the isolated domains. We hypothesized that the solubility of heterologously expressed SUR2A NBDs depends on the precise definition of the domain boundaries. Putative boundaries of SUR2A NBD1 were identified by structure-based sequence alignments and subsequently tested by exploring the solubility of SUR2A NBD1 constructs with different N and C termini. We have determined boundaries of SUR2A NBD1 that allow for soluble heterologous expression of the protein, producing a folded domain with ATP binding activity. Surprisingly, our alignment and screening data indicate that SUR2A NBD1 contains two putative, previously unidentified, regulatory elements: a large insert within the ß-sheet subdomain and a C-terminal extension. Our approach, which combines the use of structure-based sequence alignments and predictions of disordered regions combined with biochemical and biophysical studies, may be applied as a general method for developing suitable constructs of other NBDs of ABC proteins.

Physiological characterization and light response of the CO2-concentrating mechanism in the filamentous cyanobacterium Leptolyngbya sp. CPCC 696.

We studied the interactions of the CO(2)-concentrating mechanism and variable light in the filamentous cyanobacterium Leptolyngbya sp. CPCC 696 acclimated to low light (15 µmol m(-2) s(-1) PPFD) and low inorganic carbon (50 µM Ci). Mass spectrometric and polarographic analysis revealed that mediated CO(2) uptake along with both active Na(+)-independent and Na(+)-dependent HCO(3)(-) transport, likely through Na(+)/HCO(3)(-) symport, were employed to concentrate Ci internally. Combined transport of CO(2) and HCO(3)(-) required about 30 kJ mol(-1) of energy from photosynthetic electron transport to support an intracellular Ci accumulation 550-fold greater than the external Ci. Initially, Leptolyngbya rapidly induced oxygen evolution and Ci transport to reach 40-50% of maximum values by 50 µmol m(-2) s(-1) PPFD. Thereafter, photosynthesis and Ci transport increased gradually to saturation around 1,800 µmol m(-2) s(-1) PPFD. Leptolyngbya showed a low intrinsic susceptibility to photoinhibition of oxygen evolution up to PPFD of 3,000 µmol m(-2) s(-1). Intracellular Ci accumulation showed a lag under low light but then peaked at about 500 µmol photons m(-2) s(-1) and remained high thereafter. Ci influx was accompanied by a simultaneous, light-dependent, outward flux of CO(2) and by internal CO(2)/HCO(3)(-) cycling. The high-affinity and high-capacity CCM of Leptolyngbya responded dynamically to fluctuating PPFD and used excitation energy in excess of the needs of CO(2) fixation by increasing Ci transport, accumulation and Ci cycling. This capacity may allow Leptolyngbya to tolerate periodic exposure to excess high light by consuming electron equivalents and keeping PSII open.