A Potential Citrate Shunt in Erythrocytes of PKAN Patients Caused by Mutations in Pantothenate Kinase 2.

Pantothenate kinase-associated neurodegeneration (PKAN) is a progressive neurodegenerative disease caused by mutations in the pantothenate kinase 2 (PANK2) gene and associated with iron deposition in basal ganglia. Pantothenate kinase isoforms catalyze the first step in coenzyme A (CoA) biosynthesis. Since PANK2 is the only isoform in erythrocytes, these cells are an excellent ex vivo model to study the effect of PANK2 point mutations on expression/stability and activity of the protein as well as on the downstream molecular consequences. PKAN erythrocytes containing the T528M PANK2 mutant had residual enzyme activities but variable PANK2 abundances indicating an impaired regulation of the protein. Patients with G521R/G521R, G521R/G262R, and R264N/L275fs PANK2 mutants had no residual enzyme activity and strongly reduced PANK2 abundance. G521R inactivates the catalytic activity of the enzyme, whereas G262R and the R264N point mutations impair the switch from the inactive to the active conformation of the PANK2 dimer. Metabolites in cytosolic extracts were analyzed by gas chromatography-mass spectrometry and multivariate analytic methods revealing changes in the carboxylate metabolism of erythrocytes from PKAN patients as compared to that of the carrier and healthy control. Assuming low/absent CoA levels in PKAN erythrocytes, changes are consistent with a model of altered citrate channeling where citrate is preferentially converted to α-ketoglutarate and α-hydroxyglutarate instead of being used for de novo acetyl-CoA generation. This finding hints at the importance of carboxylate metabolism in PKAN pathology with potential links to reduced cytoplasmic acetyl-CoA levels in neurons and to aberrant brain iron regulation.

Impact of the dynamics of the catalytic arginine on nitrite and chlorite binding by dimeric chlorite dismutase.

Chlorite dismutases (Clds) are heme b containing oxidoreductases able to decompose chlorite to chloride and molecular oxygen. This work analyses the impact of the distal, flexible and catalytic arginine on the binding of anionic angulate ligands like nitrite and the substrate chlorite. Dimeric Cld from Cyanothece sp. PCC7425 was used as a model enzyme. We have investigated wild-type CCld having the distal catalytic R127 hydrogen-bonded to glutamine Q74 and variants with R127 (i) being arrested in a salt-bridge with a glutamate (Q74E), (ii) being fully flexible (Q74V) or (iii) substituted by either alanine (R127A) or lysine (R127K). We present the electronic and spectral signatures of the high-spin ferric proteins and the corresponding low-spin nitrite complexes elucidated by UV-visible, circular dichroism and electron paramagnetic resonance spectroscopies. Furthermore, we demonstrate the impact of the dynamics of R127 on the thermal stability of the respective nitrite adducts and present the X-ray crystal structures of the nitrite complexes of wild-type CCld and the variants Q74V, Q74E and R127A. In addition, the molecular dynamics (MD) and the binding modi of nitrite and chlorite to the ferric wild-type enzyme and the mutant proteins and the interaction of the oxoanions with R127 have been analysed by MD simulations. The findings are discussed with respect to the role(s) of R127 in ligand and chlorite binding and substrate degradation.

Order from disorder in the sarcomere: FATZ forms a fuzzy but tight complex and phase-separated condensates with α-actinin.

In sarcomeres, α-actinin cross-links actin filaments and anchors them to the Z-disk. FATZ (filamin-, α-actinin-, and telethonin-binding protein of the Z-disk) proteins interact with α-actinin and other core Z-disk proteins, contributing to myofibril assembly and maintenance. Here, we report the first structure and its cellular validation of α-actinin-2 in complex with a Z-disk partner, FATZ-1, which is best described as a conformational ensemble. We show that FATZ-1 forms a tight fuzzy complex with α-actinin-2 and propose an interaction mechanism via main molecular recognition elements and secondary binding sites. The obtained integrative model reveals a polar architecture of the complex which, in combination with FATZ-1 multivalent scaffold function, might organize interaction partners and stabilize α-actinin-2 preferential orientation in Z-disk. Last, we uncover FATZ-1 ability to phase-separate and form biomolecular condensates with α-actinin-2, raising the question whether FATZ proteins can create an interaction hub for Z-disk proteins through membraneless compartmentalization during myofibrillogenesis.

Arresting the Catalytic Arginine in Chlorite Dismutases: Impact on Heme Coordination, Thermal Stability, and Catalysis.

Chlorite dismutases (Clds) are heme b-containing oxidoreductases that can decompose chlorite to chloride and molecular oxygen. They are divided in two clades that differ in oligomerization, subunit architecture, and the hydrogen-bonding network of the distal catalytic arginine, which is proposed to switch between two conformations during turnover. To understand the impact of the conformational dynamics of this basic amino acid on heme coordination, structure, and catalysis, Cld from Cyanothece sp. PCC7425 was used as a model enzyme. As typical for a clade 2 Cld, its distal arginine 127 is hydrogen-bonded to glutamine 74. The latter has been exchanged with either glutamate (Q74E) to arrest R127 in a salt bridge or valine (Q74V) that mirrors the setting in clade 1 Clds. We present the X-ray crystal structures of Q74V and Q74E and demonstrate the pH-induced changes in the environment and coordination of the heme iron by ultraviolet-visible, circular dichroism, and electron paramagnetic resonance spectroscopies as well as differential scanning calorimetry. The conformational dynamics of R127 is shown to have a significant role in heme coordination during the alkaline transition and in the thermal stability of the heme cavity, whereas its impact on the catalytic efficiency of chlorite degradation is relatively small. The findings are discussed with respect to (i) the flexible loop connecting the N-terminal and C-terminal ferredoxin-like domains, which differs in clade 1 and clade 2 Clds and carries Q74 in clade 2 proteins, and (ii) the proposed role(s) of the arginine in catalysis.

Initial Steps to Engineer Coproheme Decarboxylase to Obtain Stereospecific Monovinyl, Monopropionyl Deuterohemes.

The oxidative decarboxylation of coproheme to form heme b by coproheme decarboxylase is a stereospecific two-step reaction. In the first step, the propionate at position two (p2) is cleaved off the pyrrole ring A to form a vinyl group at this position. Subsequently, the propionate at position four (p4) on pyrrole ring B is cleaved off and heme b is formed. In this study, we attempted to engineer coproheme decarboxylase from Corynebacterium diphtheriae to alter the stereospecificity of this reaction. By introducing a tyrosine residue in proximity to the propionate at position 4, we were able to create a new radical center in the active site. However, the artificial Tyr183• radical could not be shown to catalyze any decarboxylation.

In-depth interrogation of protein thermal unfolding data with MoltenProt.

Protein stability is a key factor in successful structural and biochemical research. However, the approaches for systematic comparison of protein stability are limited by sample consumption or compatibility with sample buffer components. Here we describe how miniaturized measurement of intrinsic tryptophan fluorescence (NanoDSF assay) in combination with a simplified description of protein unfolding can be used to interrogate the stability of a protein sample. We demonstrate that improved protein stability measures, such as apparent Gibbs free energy of unfolding, rather than melting temperature Tm , should be used to rank the results of thermostability screens. The assay is compatible with protein samples of any composition, including protein complexes and membrane proteins. Our data analysis software, MoltenProt, provides an easy and robust way to perform characterization of multiple samples. Potential applications of MoltenProt and NanoDSF include buffer and construct optimization for X-ray crystallography and cryo-electron microscopy, screening for small-molecule binding partners and comparison of effects of point mutations.

PKAN neurodegeneration and residual PANK2 activities in patient erythrocytes.

OBJECTIVE: Pantothenate kinase 2-associated neurodegeneration (PKAN) is a rare neurodegenerative disease caused by mutations in the pantothenate kinase 2 (PANK2) gene. PKAN is associated with iron deposition in the basal ganglia and, occasionally, with the occurrence of misshaped erythrocytes (acanthocytes). The aim of this study was to assess residual activity of PANK2 in erythrocytes of PKAN patients and to correlate these data with the type of PANK2 mutations and the progression of neurodegeneration. METHODS: Residual PANK2 activities in erythrocytes of 14 PKAN patients and 14 related carriers were assessed by a radiometric assay. Clinical data on neurodegeneration included the Barry-Albright Dystonia Scale (BAD-Scale) besides further general patient features. A molecular visualization and analysis program was used to rationalize the influence of the PKAN causing mutations on a molecular level. RESULTS: Erythrocytes of PKAN patients had markedly reduced or no PANK2 activity. However, patients with at least one allele of the c.1583C > T (T528M) or the c.833G > T (R278L) variant exhibited 12-56% of residual PANK2 activity. In line, molecular modeling indicated only minor effects on enzyme structure for these point mutations. On average, these patients with c.1583C > T or c.833G > T variant had lower BAD scores corresponding to lower symptom severity than patients with other PANK2 point mutations. INTERPRETATION: Residual erythrocyte PANK2 activity could be a predictor for the progression of neurodegeneration in PKAN patients. Erythrocytes are an interesting patient-derived cell system with still underestimated diagnostic potential.

A conformation-specific ON-switch for controlling CAR T cells with an orally available drug.

Molecular ON-switches in which a chemical compound induces protein-protein interactions can allow cellular function to be controlled with small molecules. ON-switches based on clinically applicable compounds and human proteins would greatly facilitate their therapeutic use. Here, we developed an ON-switch system in which the human retinol binding protein 4 (hRBP4) of the lipocalin family interacts with engineered hRBP4 binders in a small molecule-dependent manner. Two different protein scaffolds were engineered to bind to hRBP4 when loaded with the orally available small molecule A1120. The crystal structure of an assembled ON-switch shows that the engineered binder specifically recognizes the conformational changes induced by A1120 in two loop regions of hRBP4. We demonstrate that this conformation-specific ON-switch is highly dependent on the presence of A1120, as demonstrated by an ∼500-fold increase in affinity upon addition of the small molecule drug. Furthermore, the ON-switch successfully regulated the activity of primary human CAR T cells in vitro. We anticipate that lipocalin-based ON-switches have the potential to be broadly applied for the safe pharmacological control of cellular therapeutics.

HspB1 phosphorylation regulates its intramolecular dynamics and mechanosensitive molecular chaperone interaction with filamin C.

Mechanical force-induced conformational changes in proteins underpin a variety of physiological functions, typified in muscle contractile machinery. Mutations in the actin-binding protein filamin C (FLNC) are linked to musculoskeletal pathologies characterized by altered biomechanical properties and sometimes aggregates. HspB1, an abundant molecular chaperone, is prevalent in striated muscle where it is phosphorylated in response to cues including mechanical stress. We report the interaction and up-regulation of both proteins in three mouse models of biomechanical stress, with HspB1 being phosphorylated and FLNC being localized to load-bearing sites. We show how phosphorylation leads to increased exposure of the residues surrounding the HspB1 phosphosite, facilitating their binding to a compact multidomain region of FLNC proposed to have mechanosensing functions. Steered unfolding of FLNC reveals that its extension trajectory is modulated by the phosphorylated region of HspB1. This may represent a posttranslationally regulated chaperone-client protection mechanism targeting over-extension during mechanical stress.

Roles of distal aspartate and arginine of B-class dye-decolorizing peroxidase in heterolytic hydrogen peroxide cleavage.

Dye-decolorizing peroxidases (DyPs) represent the most recently classified hydrogen peroxide-dependent heme peroxidase family. Although widely distributed with more than 5000 annotated genes and hailed for their biotechnological potential, detailed biochemical characterization of their reaction mechanism remains limited. Here, we present the high-resolution crystal structures of WT B-class DyP from the pathogenic bacterium Klebsiella pneumoniae (KpDyP) (1.6 Å) and the variants D143A (1.3 Å), R232A (1.9 Å), and D143A/R232A (1.1 Å). We demonstrate the impact of elimination of the DyP-typical, distal residues Asp-143 and Arg-232 on (i) the spectral and redox properties, (ii) the kinetics of heterolytic cleavage of hydrogen peroxide, (iii) the formation of the low-spin cyanide complex, and (iv) the stability and reactivity of an oxoiron(IV)porphyrin π-cation radical (Compound I). Structural and functional studies reveal that the distal aspartate is responsible for deprotonation of H2O2 and for the poor oxidation capacity of Compound I. Elimination of the distal arginine promotes a collapse of the distal heme cavity, including blocking of one access channel and a conformational change of the catalytic aspartate. We also provide evidence of formation of an oxoiron(IV)-type Compound II in KpDyP with absorbance maxima at 418, 527, and 553 nm. In summary, a reaction mechanism of the peroxidase cycle of B-class DyPs is proposed. Our observations challenge the idea that peroxidase activity toward conventional aromatic substrates is related to the physiological roles of B-class DyPs.

Secreted heme peroxidase from Dictyostelium discoideum: Insights into catalysis, structure, and biological role.

Oxidation of halides and thiocyanate by heme peroxidases to antimicrobial oxidants is an important cornerstone in the innate immune system of mammals. Interestingly, phylogenetic and physiological studies suggest that homologous peroxidases are already present in mycetozoan eukaryotes such as Dictyostelium discoideum This social amoeba kills bacteria via phagocytosis for nutrient acquisition at its single-cell stage and for antibacterial defense at its multicellular stages. Here, we demonstrate that peroxidase A from D. discoideum (DdPoxA) is a stable, monomeric, glycosylated, and secreted heme peroxidase with homology to mammalian peroxidases. The first crystal structure (2.5 Å resolution) of a mycetozoan peroxidase of this superfamily shows the presence of a post-translationally-modified heme with one single covalent ester bond between the 1-methyl heme substituent and Glu-236. The metalloprotein follows the halogenation cycle, whereby compound I oxidizes iodide and thiocyanate at high rates (>108 m-1 s-1) and bromide at very low rates. It is demonstrated that DdPoxA is up-regulated and likely secreted at late multicellular development stages of D. discoideum when migrating slugs differentiate into fruiting bodies that contain persistent spores on top of a cellular stalk. Expression of DdPoxA is shown to restrict bacterial contamination of fruiting bodies. Structure and function of DdPoxA are compared with evolutionary-related mammalian peroxidases in the context of non-specific immune defense.

Molecular Mechanism of Enzymatic Chlorite Detoxification: Insights from Structural and Kinetic Studies.

The heme enzyme chlorite dismutase (Cld) catalyzes the degradation of chlorite to chloride and dioxygen. Although structure and steady-state kinetics of Clds have been elucidated, many questions remain (e.g., the mechanism of chlorite cleavage and the pH dependence of the reaction). Here, we present high-resolution X-ray crystal structures of a dimeric Cld at pH 6.5 and 8.5, its fluoride and isothiocyanate complexes and the neutron structure at pH 9.0 together with the pH dependence of the Fe(III)/Fe(II) couple, and the UV-vis and resonance Raman spectral features. We demonstrate that the distal Arg127 cannot act as proton acceptor and is fully ionized even at pH 9.0 ruling out its proposed role in dictating the pH dependence of chlorite degradation. Stopped-flow studies show that (i) Compound I and hypochlorite do not recombine and (ii) Compound II is the immediately formed redox intermediate that dominates during turnover. Homolytic cleavage of chlorite is proposed.

Two-faced Fcab prevents polymerization with VEGF and reveals thermodynamics and the 2.15 Å crystal structure of the complex.

Fcabs (Fc domain with antigen-binding sites) are promising novel therapeutics. By engineering of the C-terminal loops of the CH3 domains, 2 antigen binding sites can be inserted in close proximity. To elucidate the binding mode(s) between homodimeric Fcabs and small homodimeric antigens, the interaction between the Fcabs 448 and CT6 (having the AB, CD and EF loops and the C-termini engineered) with homodimeric VEGF was investigated. The crystal structures of these Fcabs, which form polymers with the antigen VEGF in solution, were determined. However, construction of heterodimeric Fcabs (JanusFcabs: one chain Fc-wt, one chain VEGF-binding) results in formation of distinct JanusFcab-VEGF complexes (2:1), which allowed elucidation of the crystal structure of the JanusCT6-VEGF complex at 2.15 Å resolution. VEGF binding to Janus448 and JanusCT6 is shown to be entropically unfavorable, but enthalpically favorable. Structure-function relationships are discussed with respect to Fcab design and engineering strategies.

Fcab-HER2 Interaction: a Ménage à Trois. Lessons from X-Ray and Solution Studies.

The crystallizable fragment (Fc) of the immunoglobulin class G (IgG) is an attractive scaffold for the design of novel therapeutics. Upon engineering the C-terminal loops in the CH3 domains, Fcabs (Fc domain with antigen-binding sites) can be designed. We present the first crystal structures of Fcabs, i.e., of the HER2-binding clone H10-03-6 having the AB and EF loop engineered and the stabilized version STAB19 derived by directed evolution. Comparison with the crystal structure of the Fc wild-type protein reveals conservation of the overall domain structures but significant differences in EF-loop conformations. Furthermore, we present the first Fcab-antigen complex structures demonstrating the interaction between the engineered Fcab loops with domain IV of HER2. The crystal structures of the STAB19-HER2 and H10-03-6-HER2 complexes together with analyses by isothermal titration calorimetry, SEC-MALS, and fluorescence correlation spectroscopy show that one homodimeric Fcab binds two HER2 molecules following a negative cooperative binding behavior.

Hydrogen peroxide-mediated conversion of coproheme to heme b by HemQ-lessons from the first crystal structure and kinetic studies.

Heme biosynthesis in Gram-positive bacteria follows a recently described coproporphyrin-dependent pathway with HemQ catalyzing the decarboxylation of coproheme to heme b. Here we present the first crystal structure of a HemQ (homopentameric coproheme-HemQ from Listeria monocytogenes) at 1.69 Å resolution and the conversion of coproheme to heme b followed by UV-vis and resonance Raman spectroscopy as well as mass spectrometry. The ferric five-coordinated coproheme iron of HemQ is weakly bound by a neutral proximal histidine H174. In the crystal structure of the resting state, the distal Q187 (conserved in Firmicutes HemQ) is H-bonded with propionate p2 and the hydrophobic distal cavity lacks solvent water molecules. Two H2 O2 molecules are shown to be necessary for decarboxylation of the propionates p2 and p4, thereby forming the corresponding vinyl groups of heme b. The overall reaction is relatively slow (kcat /KM = 1.8 × 102 m-1 ·s-1 at pH 7.0) and occurs in a stepwise manner with a three-propionate intermediate. We present the noncovalent interactions between coproheme and the protein and propose a two-step reaction mechanism. Furthermore, the structure of coproheme-HemQ is compared to that of the phylogenetically related heme b-containing chlorite dismutases. DATABASE: Structural data are available in the PDB under the accession number 5LOQ.

Chemistry and Molecular Dynamics Simulations of Heme b-HemQ and Coproheme-HemQ.

Recently, a novel pathway for heme b biosynthesis in Gram-positive bacteria has been proposed. The final poorly understood step is catalyzed by an enzyme called HemQ and includes two decarboxylation reactions leading from coproheme to heme b. Coproheme has been suggested to act as both substrate and redox active cofactor in this reaction. In the study presented here, we focus on HemQs from Listeria monocytogenes (LmHemQ) and Staphylococcus aureus (SaHemQ) recombinantly produced as apoproteins in Escherichia coli. We demonstrate the rapid and two-phase uptake of coproheme by both apo forms and the significant differences in thermal stability of the apo forms, coproheme-HemQ and heme b-HemQ. Reduction of ferric high-spin coproheme-HemQ to the ferrous form is shown to be enthalpically favored but entropically disfavored with standard reduction potentials of -205 ± 3 mV for LmHemQ and -207 ± 3 mV for SaHemQ versus the standard hydrogen electrode at pH 7.0. Redox thermodynamics suggests the presence of a pronounced H-bonding network and restricted solvent mobility in the heme cavity. Binding of cyanide to the sixth coproheme position is monophasic but relatively slow (∼1 × 10(4) M(-1) s(-1)). On the basis of the available structures of apo-HemQ and modeling of both loaded forms, molecular dynamics simulation allowed analysis of the interaction of coproheme and heme b with the protein as well as the role of the flexibility at the proximal heme cavity and the substrate access channel for coproheme binding and heme b release. Obtained data are discussed with respect to the proposed function of HemQ in monoderm bacteria.

From chlorite dismutase towards HemQ - the role of the proximal H-bonding network in haeme binding.

Chlorite dismutase (Cld) and HemQ are structurally and phylogenetically closely related haeme enzymes differing fundamentally in their enzymatic properties. Clds are able to convert chlorite into chloride and dioxygen, whereas HemQ is proposed to be involved in the haeme b synthesis of Gram-positive bacteria. A striking difference between these protein families concerns the proximal haeme cavity architecture. The pronounced H-bonding network in Cld, which includes the proximal ligand histidine and fully conserved glutamate and lysine residues, is missing in HemQ. In order to understand the functional consequences of this clearly evident difference, specific hydrogen bonds in Cld from 'Candidatus Nitrospira defluvii' (NdCld) were disrupted by mutagenesis. The resulting variants (E210A and K141E) were analysed by a broad set of spectroscopic (UV-vis, EPR and resonance Raman), calorimetric and kinetic methods. It is demonstrated that the haeme cavity architecture in these protein families is very susceptible to modification at the proximal site. The observed consequences of such structural variations include a significant decrease in thermal stability and also affinity between haeme b and the protein, a partial collapse of the distal cavity accompanied by an increased percentage of low-spin state for the E210A variant, lowered enzymatic activity concomitant with higher susceptibility to self-inactivation. The high-spin (HS) ligand fluoride is shown to exhibit a stabilizing effect and partially restore wild-type Cld structure and function. The data are discussed with respect to known structure-function relationships of Clds and the proposed function of HemQ as a coprohaeme decarboxylase in the last step of haeme biosynthesis in Firmicutes and Actinobacteria.

Dimeric chlorite dismutase from the nitrogen-fixing cyanobacterium Cyanothece sp. PCC7425.

It is demonstrated that cyanobacteria (both azotrophic and non-azotrophic) contain heme b oxidoreductases that can convert chlorite to chloride and molecular oxygen (incorrectly denominated chlorite 'dismutase', Cld). Beside the water-splitting manganese complex of photosystem II, this metalloenzyme is the second known enzyme that catalyses the formation of a covalent oxygen-oxygen bond. All cyanobacterial Clds have a truncated N-terminus and are dimeric (i.e. clade 2) proteins. As model protein, Cld from Cyanothece sp. PCC7425 (CCld) was recombinantly produced in Escherichia coli and shown to efficiently degrade chlorite with an activity optimum at pH 5.0 [kcat 1144 ± 23.8 s(-1), KM 162 ± 10.0 µM, catalytic efficiency (7.1 ± 0.6) × 10(6) M(-1) s(-1)]. The resting ferric high-spin axially symmetric heme enzyme has a standard reduction potential of the Fe(III)/Fe(II) couple of -126 ± 1.9 mV at pH 7.0. Cyanide mediates the formation of a low-spin complex with k(on) = (1.6 ± 0.1) × 10(5) M(-1) s(-1) and k(off) = 1.4 ± 2.9 s(-1) (KD ∼ 8.6 µM). Both, thermal and chemical unfolding follows a non-two-state unfolding pathway with the first transition being related to the release of the prosthetic group. The obtained data are discussed with respect to known structure-function relationships of Clds. We ask for the physiological substrate and putative function of these O2 -producing proteins in (nitrogen-fixing) cyanobacteria.

Structure and heme-binding properties of HemQ (chlorite dismutase-like protein) from Listeria monocytogenes.

Chlorite dismutase-like proteins are structurally closely related to functional chlorite dismutases which are heme b-dependent oxidoreductases capable of reducing chlorite to chloride with simultaneous production of dioxygen. Chlorite dismutase-like proteins are incapable of performing this reaction and their biological role is still under discussion. Recently, members of this large protein family were shown to be involved in heme biosynthesis in Gram-positive bacteria, and thus the protein was renamed HemQ in these organisms. In the present work the structural and heme binding properties of the chlorite dismutase-like protein from the Gram-positive pathogen Listeria monocytogenes (LmCld) were analyzed in order to evaluate its potential role as a regulatory heme sensing protein. The homopentameric crystal structure (2.0Å) shows high similarity to chlorite-degrading chlorite dismutases with an important difference in the structure of the putative substrate and heme entrance channel. In solution LmCld is a stable hexamer able to bind the low-spin ligand cyanide. Heme binding is reversible with KD-values determined to be 7.2µM (circular dichroism spectroscopy) and 16.8µM (isothermal titration calorimetry) at pH 7.0. Both acidic and alkaline conditions promote heme release. Presented biochemical and structural data reveal that the chlorite dismutase-like protein from L. monocytogenes could act as a potential regulatory heme sensing and storage protein within heme biosynthesis.

The Center for Optimized Structural Studies (COSS) platform for automation in cloning, expression, and purification of single proteins and protein-protein complexes.

Expression in Escherichia coli represents the simplest and most cost effective means for the production of recombinant proteins. This is a routine task in structural biology and biochemistry where milligrams of the target protein are required in high purity and monodispersity. To achieve these criteria, the user often needs to screen several constructs in different expression and purification conditions in parallel. We describe a pipeline, implemented in the Center for Optimized Structural Studies, that enables the systematic screening of expression and purification conditions for recombinant proteins and relies on a series of logical decisions. We first use bioinformatics tools to design a series of protein fragments, which we clone in parallel, and subsequently screen in small scale for optimal expression and purification conditions. Based on a scoring system that assesses soluble expression, we then select the top ranking targets for large-scale purification. In the establishment of our pipeline, emphasis was put on streamlining the processes such that it can be easily but not necessarily automatized. In a typical run of about 2 weeks, we are able to prepare and perform small-scale expression screens for 20-100 different constructs followed by large-scale purification of at least 4-6 proteins. The major advantage of our approach is its flexibility, which allows for easy adoption, either partially or entirely, by any average hypothesis driven laboratory in a manual or robot-assisted manner.