Adam Pomorski

Exploration of Biarsenical Chemistry—Challenges in Protein Research

The fluorescent modification of proteins (with genetically encoded low‐molecular‐mass fluorophores, affinity probes, or other chemically active species) is extraordinarily useful for monitoring and controlling protein functions in vitro, as well as in cell cultures and tissues. The large sizes of some fluorescent tags, such as fluorescent proteins, often perturb normal activity and localization of the protein of interest, as well as other effects. Of the many fluorescent‐labeling strategies applied to in vitro and in vivo studies, one is very promising. This requires a very short (6‐ to12‐residue), appropriately spaced, tetracysteine sequence (CCXXCC); this is either placed at a protein terminus, within flexible loops, or incorporated into secondary structure elements. Proteins that contain the tetracysteine motif become highly fluorescent upon labeling with a nonluminescent biarsenical probe, and form very stable covalent complexes. We focus on the development, growth, and multiple applications of this protein research methodology, both in vitro and in vivo. Its application is not limited to intact‐cell protein visualization; it has tremendous potential in other protein research disciplines, such as protein purification and activity control, electron microscopy imaging of cells or tissue, protein–protein interaction studies, protein stability, and aggregation studies.

Molar absorption coefficients and stability constants of metal complexes of 4-(2-pyridylazo)resorcinol (PAR): Revisiting common chelating probe for the study of metalloproteins

4-(2-Pyridylazo)resorcinol (PAR) is one of the most popular chromogenic chelator used in the determination of the concentrations of various metal ions from the d, p and f blocks and their affinities for metal ion-binding biomolecules. The most important characteristics of such a sensor are the molar absorption coefficient and the metal-ligand complex dissociation constant. However, it must be remembered that these values are dependent on the specific experimental conditions (e.g. pH, solvent components, and reactant ratios). If one uses these values to process data obtained in different conditions, the final result can be under- or overestimated. We aimed to establish the spectral properties and the stability of PAR and its complexes accurately with Zn(2+), Cd(2+), Hg(2+), Co(2+), Ni(2+), Cu(2+), Mn(2+) and Pb(2+) at a multiple pH values. The obtained results account for the presence of different species of metal-PAR complexes in the physiological pH range of 5 to 8 and have been frequently neglected in previous studies. The effective molar absorption coefficient at 492 nm for the ZnHx(PAR)2 complex at pH7.4 in buffered water solution is 71,500 M(-1) cm(-1), and the dissociation constant of the complex in these conditions is 7.08×10(-13) M(2). To confirm these values and estimate the range of the dissociation constants of zinc-binding biomolecules that can be measured using PAR, we performed several titrations of zinc finger peptides and zinc chelators. Taken together, our results provide the updated parameters that are applicable to any experiment conducted using inexpensive and commercially available PAR.

Method for accurate determination of dissociation constants of optical ratiometric systems: chemical probes, genetically encoded sensors, and interacting molecules.

Ratiometric chemical probes and genetically encoded sensors are of high interest for both analytical chemists and molecular biologists. Their high sensitivity toward the target ligand and ability to obtain quantitative results without a known sensor concentration have made them a very useful tool in both in vitro and in vivo assays. Although ratiometric sensors are widely used in many applications, their successful and accurate usage depends on how they are characterized in terms of sensing target molecules. The most important feature of probes and sensors besides their optical parameters is an affinity constant toward analyzed molecules. The literature shows that different analytical approaches are used to determine the stability constants, with the ratio approach being most popular. However, oversimplification and lack of attention to detail results in inaccurate determination of stability constants, which in turn affects the results obtained using these sensors. Here, we present a new method where ratio signal is calibrated for borderline values of intensities of both wavelengths, instead of borderline ratio values that generate errors in many studies. At the same time, the equation takes into account the cooperativity factor or fluorescence artifacts and therefore can be used to characterize systems with various stoichiometries and experimental conditions. Accurate determination of stability constants is demonstrated utilizing four known optical ratiometric probes and sensors, together with a discussion regarding other, currently used methods.

The High ZnII Affinity of the Tetracysteine Tag Affects Its Fluorescent Labeling with Biarsenicals

Fluorescent labeling of proteins is one of the most popular and applicable methods in molecular diagnostic studies used for quantification assays, as well as for determination of protein–protein or ligand–protein interactions. 2] The imaging of the labeled proteins is of great importance for the understanding of protein function, intraor intercellular distribution of proteins, and sensing of biological events. Genetically encoded sensors derived from fluorescent proteins (GFP and others) are extremely useful for those studies; however, the large size and spectral properties of fluorescent proteins introduce certain limitations for their applications. In the last decade, researchers successfully imaged proteins in cells by using biarsenical fluorescein (FlAsH-EDT2) developed by R. Y. Tsien and co-workers. [3]

Molar absorption coefficients and stability constants of Zincon metal complexes for determination of metal ions and bioinorganic applications

Zincon (ZI) is one of the most common chromophoric chelating probes for the determination of Zn2+ and Cu2+ ions. It is also known to bind other metal ions. However, literature data on its binding properties and molar absorption coefficients are rather poor, varying among publications or determined only in certain conditions. There are no systematic studies on Zn2+ and Cu2+ affinities towards ZI performed under various conditions. However, this widely commercially available and inexpensive agent is frequently the first choice probe for the measurement of metal binding and release as well as determination of affinity constants of other ligands/macromolecules of interest. Here, we establish the spectral properties and the stability of ZI and its complexes with Zn2+, Cu2+, Cd2+, Hg2+, Co2+, Ni2+ and Pb2+ at multiple pH values from 6 to 9.9. The obtained results show that in water solution the MZI complex is predominant, but in the case of Co2+ and Ni2+, M(ZI)2 complexes are also formed. The molar absorption coefficient at 618 nm for ZnZI and 599nm for CuZI complexes at pH7.4 in buffered (I=0.1M) water solutions are 24,200 and 26,100M-1cm-1, respectively. Dissociation constants of those complexes are 2.09×10-6 and 4.68×10-17M. We also characterized the metal-assisted Zincon decomposition. Our results provide new and reassessed optical and stability data that are applicable to a wide range of chemical and bioinorganic applications including metal ion detection, and quantification and affinity studies of ligands of interest. SYNOPSIS Accurate values of molar absorption coefficients of Zincon complex with Zn2+, Cd2+, Hg2+, Co2+, Ni2+, Cu2+, and Pb2+ for rapid metal ion quantification are provided. Zincon stability constants with Zn2+ and Cu2+ in a wide pH range were determined.

Revisiting Mitochondrial pH with an Improved Algorithm for Calibration of the Ratiometric 5(6)-carboxy-SNARF-1 Probe Reveals Anticooperative Reaction with H+ Ions and Warrants Further Studies of Organellar pH.

Fluorescence measurements of pH and other analytes in the cell rely on accurate calibrations, but these have routinely used algorithms that inadequately describe the properties of indicators. Here, we have established a more accurate method for calibrating and analyzing data obtained using the ratiometric probe 5(6)-carboxy-SNARF-1. We tested the implications of novel approach to measurements of pH in yeast mitochondria, a compartment containing a small number of free H+ ions. Our findings demonstrate that 5(6)-carboxy-SNARF-1 interacts with H+ ions inside the mitochondria in an anticooperative manner (Hill coefficient n of 0.5) and the apparent pH inside the mitochondria is ~0.5 unit lower than had been generally assumed. This result, at odds with the current consensus on the mechanism of energy generation in the mitochondria, is in better agreement with theoretical considerations and warrants further studies of organellar pH.

Short-sweep capillary electrophoresis with a selective zinc fluorescence imaging reagent FluoZin-3 for determination of free and metalothionein-2a-bound Zn 2+ ions

A capillary electrophoretic (CE) method using a short-sweep approach and laser-induced fluorescence (LIF) detection (ShortSweepCE-LIF) was developed for determination of Zn2+ and Cd2+ as complexes with highly selective and sensitive fluorescent probe FluoZin-3. The ShortSweepCE-LIF method, established in this work, can be used for examining competitive Zn2+ and Cd2+ binding properties of metalloproteins or peptides. The parameters including background electrolyte composition, injection pressure and time as well as separation voltage were investigated. Under the optimized conditions, 80 mM HEPES, pH 7.4, with 1.5 μM FluoZin-3 was used as an electrolyte, hydrodynamic injection was performed at 50 mbar for 5 s, and separation voltage of 25 kV. Limits of detection for Zn2+ and Cd2+ were 4 and 125 nM, respectively. The developed method was demonstrated in a study of interactions between metalothionein-2a isoform and metal ions Zn2+, Co2+ and Cd2+. It was found that FluoZin-3 was able to extract a single Zn2+ ion, while added Co2+ (in surplus) extracted only 2.4 Zn2+ ions, and Cd2+ extracted all 7 Zn2+ ions present in the metalothionein molecule.

Fluorescent probes for selective protein labeling in lysosomes: a case of α-galactosidase A

Fluorescence‐based live‐cell imaging (LCI) of lysosomal glycosidases is often hampered by unfavorable pH and redox conditions that reduce fluorescence output. Moreover, most lysosomal glycosidases are low‐mass soluble proteins that do not allow for bulky fluorescent protein fusions. We selected a‐galactosidase A (GALA) as a model lysosomal glycosidase involved in Anderson‐Fabry disease (AFD) for the current LCI approach. Examination of the subcellular localization of AFD‐causing mutants can reveal the mechanism underlying cellular trafficking deficits. To minimize genetic GALA modification, we employed a biarsenical labeling protocol with tetracysteine (TC‐tag) detection. We tested the efficiency of halogen‐substituted biarsenical probes to interact with C‐terminally TC‐tagged GALA peptide at pH 4.5 in vitro and identified F2FlAsH‐EDT2 as a superior detection reagent for GALA. This probe provides improved signal/noise ratio in labeled COS‐7 cells transiently expressing TC‐tagged GALA. The investigated fluorescence‐based LCI technology of TC‐tagged lysosomal protein using an improved biarsenical probe can be used to identify novel compounds that promote proper trafficking of mutant GALA to lysosomal compartments and rescue the mutant phenotype.—Bohl, C., Pomorski, A., Seemann, S., Knospe, A.‐M., Zheng, C., Kreżel, A., Rolfs, A., Lukas, J. Fluorescent probes for selective protein labeling in lysosomes: a case of α‐galactosidase A. FASEB J. 31, 5258–5267 (2017). www.fasebj.org

Optimized allosteric inhibition of engineered protein tyrosine phosphatases with an expanded palette of biarsenical small molecules

Protein tyrosine phosphatases (PTPs), which catalyze the dephosphorylation of phosphotyrosine in protein substrates, are important cell-signaling regulators, as well as potential drug targets for a range of human diseases. Chemical tools for selectively targeting the activities of individual PTPs would help to elucidate PTP signaling roles and potentially expedite the validation of PTPs as therapeutic targets. We have recently reported a novel strategy for the design of non-natural allosteric-inhibition sites in PTPs, in which a tricysteine moiety is engineered within the PTP catalytic domain at a conserved location outside of the active site. Introduction of the tricysteine motif, which does not exist in any wild-type PTP, serves to sensitize target PTPs to inhibition by a biarsenical compound, providing a generalizable strategy for the generation of allosterically sensitized (as) PTPs. Here we show that the potency, selectivity, and kinetics of asPTP inhibition can be significantly improved by exploring the inhibitory action of a range of biarsenical compounds that differ in interarsenical distance, steric bulk, and electronic structure. By investigating the inhibitor sensitivities of five asPTPs from four different subfamilies, we have found that asPTP catalytic domains can be broadly divided into two groups: one that is most potently inhibited by biarsenical compounds with large interarsenical distances, such as AsCy3-EDT2, and one that is most potently inhibited by compounds with relatively small interarsenical distances, such as FlAsH-EDT2. Moreover, we show that a tetrachlorinated derivative of FlAsH-EDT2, Cl4FlAsH-EDT2, targets asPTPs significantly more potently than the parent compound, both in vitro and in asPTP-expressing cells. Our results show that biarsenicals with altered interarsenical distances and electronic properties are important tools for optimizing the control of asPTP activity and, more broadly, suggest that diversification of biarsenical libraries can serve to increase the efficacy of these compounds in targeted control of protein function.