Secretion of mutant leucine-specific binding proteins with internal deletions in Escherichia coli.

The leucine-specific binding protein, encoded by the livK gene, is located in the periplasm of E. coli. The present study is an attempt to identify intragenic regions that determine the efficiency of its secretion into the periplasm. C-terminal deletions or fusions of the livK gene to trpA (encoding the alpha subunit of tryptophan synthetase) were secreted with little loss of efficiency [1]. A series of deletions was constructed at the unique Sphl site within livK, near the 5' end of the region coding for the mature protein. Between 16 and 113 amino acids were deleted in the amino-terminal one-third of the protein. A few of these deletions were located within a few amino acids of the signal sequence processing site. Deletions extending within thirteen residues of the processing site were processed and secreted more slowly than normal. Secondary structure predictions suggested that the alpha-helical core region of the signal sequence extends into the mature protein in the case of the slow processing mutants, perhaps interfering with the recognition site for leader peptidase or other secretory components. These results suggest that the conformation around the signal processing site may be a critical factor in determining the efficiency of secretion. During the course of this study, it was found that the difference in molecular weight between precursor and mature forms of some binding protein mutants, as judged by SDS-PAGE, was much greater than could be accounted for by processing of the signal sequence. This anomalous mobility on gels, however, could be eliminated by performing SDS-PAGE in the presence of 6 M urea.     

Exploring the conformational equilibrium of E. coli thioredoxin reductase: characterization of two catalytically important states by ultrafast flavin fluorescence spectroscopy

The conformational dynamics of wild-type Escherichia coli thioredoxin reductase (TrxR) and the mutant enzyme C138S were studied by ultrafast time-resolved fluorescence of the flavin cofactor in combination with circular dichroism (both in the flavin fingerprint and far-UV regions) and steady-state fluorescence and absorption spectroscopy. The spectroscopic data show two conformational states of the enzyme (named FO and FR), of which the physical characteristics differ considerably. Ultrafast fluorescence lifetime measurements make it possible to distinguish between the two different populations: Dominant picosecond lifetimes of approximately 1 ps (contribution 75%) and 7 ps (8%) are associated with the FO species in TrxR C138S. Long-lived fluorescence with two time constants in the range of 0.2-1 ns (total contribution 17%) originates from enzyme molecules in the FR conformation. The near absence of fast lifetime components in oxidized wild-type TrxR supports the idea of this enzyme being predominantly in the FR conformation. The emission spectrum of the FO conformation is blue-shifted with respect to that of the FR conformation. Because of the large difference in fluorescence characteristics, fluorescence measurements on time scales longer than 100 ps are fully determined by the fraction of enzyme molecules in the FR conformation. Binding of the thiol reagent phenyl mercuric acetate to wild-type enzyme and TrxR C138S stabilizes the enzymes in the FR conformation. Specific binding of the NADPH-analog, AADP(+), to the FR conformation resulted in dynamic fluorescence quenching in support of the multiple quenching sites model. Raising the temperature from 277K-323K resulted in a moderate shift to the FR conformation for TrxR C138S. High concentrations of the cosolvent glycerol triggered the domain rotation from the FO to the FR conformation.     

Identification of CRISP2 from human sperm as PSP94‐binding protein and generation of CRISP2‐specific anti‐peptide antibodies

Cysteine‐rich secretory proteins (CRISPs) are mainly found in the mammalian male reproductive tract and reported to be involved at different stages of fertilization. CRISPs have been shown to interact with prostate secretory protein of 94 amino acids (PSP94) from diverse sources, and the binding of these evolutionarily conserved proteins across species is proposed to be of functional significance. Of the three mammalian CRISPs, PSP94–CRISP3 interaction is well characterized, and specific binding sites have been identified; whereas, CRISP2 has been shown to interact with PSP94 in vitro. Interestingly, human CRISP3 and CRISP2 proteins are closely related showing 71.4% identity. In this study, we identified CRISP2 as a potential binding protein of PSP94 from human sperm. Further, we generated antisera capable of specifically detecting CRISP2 and not CRISP3. In this direction, specific peptides corresponding to the least conserved ion channel regulatory region were synthesized, and polyclonal antibodies were generated against the peptide in rabbits. The binding characteristics of the anti‐CRISP2 peptide antibody were evaluated using competitive ELISA. Immunoblotting experiments also confirmed that the peptide was able to generate antibodies capable of detecting the mature CRISP2 protein present in human sperm lysate. Furthermore, this anti‐CRISP2 peptide antibody also detected the presence of native CRISP2 on sperm.Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Δ98Δ, a minimalist model of antiparallel β‐sheet proteins based on intestinal fatty acid binding protein

The design of β‐barrels has always been a formidable challenge for de novo protein design. For instance, a persistent problem is posed by the intrinsic tendency to associate given by free edges. From the opposite standpoint provided by the redesign of natural motifs, we believe that the intestinal fatty acid binding protein (IFABP) framework allows room for intervention, giving rise to abridged forms from which lessons on β‐barrel architecture and stability could be learned. In this context, Δ98Δ (encompassing residues 29–126 of IFABP) emerges as a monomeric variant that folds properly, retaining functional activity, despite lacking extensive stretches involved in the closure of the β‐barrel. Spectroscopic probes (fluorescence and circular dichroism) support the existence of a form preserving the essential determinants of the parent structure, albeit endowed with enhanced flexibility. Chemical and physical perturbants reveal cooperative unfolding transitions, with evidence of significant population of intermediate species in equilibrium, structurally akin to those transiently observed in IFABP. The recognition by the natural ligand oleic acid exerts a mild stabilizing effect, being of a greater magnitude than that found for IFABP. In summary, Δ98Δ adopts a monomeric state with a compact core and a loose periphery, thus pointing to the nonintuitive notion that the integrity of the β‐barrel can indeed be compromised with no consequence on the ability to attain a native‐like and functional fold.     

Solution structure of the E.coli TolA C-terminal domain reveals conformational changes upon binding to the phage g3p N-terminal domain

The Tol-Pal system of Escherichia coli is a macromolecular complex located in the cell envelope. It is involved in maintaining the integrity of the outer membrane and is required for the uptake of two different types of macromolecules, which are bacteriotoxins (colicins) and DNA of filamentous bacteriophages. The TolA protein plays a central role in these import mechanisms. Its C-terminal domain (TolAIII) is involved in the translocation step via direct interaction with the N-terminal domain of colicins and the N-terminal domain of the phage minor coat gene 3 protein (g3pN1). Extreme behaviours of TolAIII have been previously observed, since the structure of TolAIII either remained unaffected or adopted disordered conformation upon binding to different pore-forming colicins. Here, we have solved the 3D structure of free TolAIII by heteronuclear NMR spectroscopy and compared it to the crystal structure of TolAIII bound to g3pN1 in order to study the effect of g3pN1 on the tertiary structure of TolAIII. Backbone 1H, 15N and 13C resonances of the g3pN1-bound TolAIII were also assigned and used to superimpose the solution structure of free TolAIII on the crystal structure of the g3pN1-TolAIII fusion protein. This allowed us to track conformational changes of TolAIII upon binding. While the global fold of free TolAIII is mainly identical to that of g3pN1-bound TolAIII, shift of secondary structures does occur. Thus, TolAIII, which interacts also in vivo with Pal and TolB, is able to adapt its conformation upon binding to various partners. Possible models for protein binding mechanisms are discussed to explain this so-far unobserved behaviour of TolAIII.     

Targeted molecular dynamics simulation studies of binding and conformational changes in E. coli MurD

Enzymes involved in the biosynthesis of bacterial peptidoglycan, an essential cell wall polymer unique to prokaryotic cells, represent a highly interesting target for antibacterial drug design. Structural studies of E. coli MurD, a three-domain ATP hydrolysis driven muramyl ligase revealed two inactive open conformations of the enzyme with a distinct C-terminal domain position. It was hypothesized that the rigid body rotation of this domain brings the enzyme to its closed active conformation, a structure, which was also determined experimentally. Targeted molecular dynamics 1 ns-length simulations were performed in order to examine the substrate binding process and gain insight into structural changes in the enzyme that occur during the conformational transitions into the active conformation. The key interactions essential for the conformational transitions and substrate binding were identified. The results of such studies provide an important step toward more powerful exploitation of experimental protein structures in structure-based inhibitor design.     

Mechanism of formation of the C‐terminal β‐hairpin of the B3 domain of the immunoglobulin binding protein G from Streptococcus. I. Importance of hydrophobic interactions in stabilization of β‐hairpin structure

We previously studied a 16‐amino acid‐residue fragment of the C‐terminal β‐hairpin of the B3 domain (residues 46–61), [IG(46–61)] of the immunoglobulin binding protein G from Streptoccocus, and found that hydrophobic interactions and the turn region play an important role in stabilizing the structure. Based on these results, we carried out systematic structural studies of peptides derived from the sequence of IG (46–61) by systematically shortening the peptide by one residue at a time from both the C‐ and the N‐terminus. To determine the structure and stability of two resulting 12‐ and 14‐amino acid‐residue peptides, IG(48–59) and IG(47–60), respectively, we carried out circular dichroism, NMR, and calorimetric studies of these peptides in pure water. Our results show that IG(48–59) possesses organized three‐dimensional structure stabilized by hydrophobic interactions (Tyr50–Phe57 and Trp48–Val59) at T = 283 and 305 K. At T = 313 K, the structure breaks down because of increased chain entropy, but the turn region is preserved in the same position observed for the structure of the whole protein. The breakdown of structure occurs near the melting temperature of this peptide (T_m = 310 K) measured by differential scanning calorimetry (DSC). The melting temperature of IG(47–60) determined by DSC is T_m = 330 K and its structure is similar to that of the native β‐hairpin at all (lower) temperatures examined (283–313 K). Both of these truncated sequences are conserved in all known amino acid sequences of the B domains of the immunoglobulin binding protein G from bacteria. Thus, this study contributes to an understanding of the mechanism of folding of this whole family of proteins, and provides information about the mechanism of formation and stabilization of a β‐hairpin structural element. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

A conformational change in the lactose permease of Escherichia coli is induced by ligand binding or membrane potential.

Lactose transport in membrane vesicles containing lactose permease with a single Cys residue in place of Val 315 is inactivated by N-ethylmaleimide in a manner that is stimulated by substrate or by a H+ electrochemical gradient (delta microH+; Sahin-Tóth M, Kaback HR, 1993, Protein Sci 2:1024-1033). The findings are confirmed and extended in this communication. Purified, reconstituted Val 315-->Cys permease reacts with N-ethylmaleimide or hydrophobic fluorescent maleimides but not with a membrane impermeant thiol reagent, and beta-galactosides specifically stimulate the rate of labeling. Furthermore, the reactivity of purified Val 315-->Cys permease is enhanced by imposition of a membrane potential (delta psi, interior negative). The results indicate that either ligand binding or delta psi induces a conformational change in the permease that brings the N-terminus of helix X into an environment that is more accessible from the lipid phase.     

2‐D DIGE reveals changes in wheat xylanase inhibitor protein families due to Fusarium graminearum ΔTri5 infection and grain development

Wheat contains three different classes of proteinaceous xylanase inhibitors (XIs), i.e. Triticum aestivum xylanase inhibitors (TAXIs) xylanase‐inhibiting proteins (XIPs), and thaumatin‐like xylanase inhibitors (TLXIs) which are believed to act as a defensive barrier against phytopathogenic attack. In the absence of relevant data in wheat kernels, we here examined the response of the different members of the XI protein population to infection with a ΔTri5 mutant of Fusarium graminearum, the wild type of which is one of the most important wheat ear pathogens, in early developing wheat grain. Wheat ears were inoculated at anthesis, analyzed using 2‐D DIGE and multivariate analysis at 5, 15, and 25 days post anthesis (DPA), and compared with control samples. Distinct abundance patterns could be distinguished for different XI forms in response to infection with F. graminearum ΔTri5. Some (iso)forms were up‐regulated, whereas others were down‐regulated. This pathogen‐specific regulation of proteins was mostly visible at five DPA and levelled off in the samples situated further from the inoculation point. Furthermore, it was shown that most identified TAXI‐ and XIP‐type XI (iso)forms significantly increased in abundance from the milky (15 DPA) to the soft dough stages (25 DPA) on a per kernel basis, although the extent of increase differed greatly. Non‐glycosylated XIP forms increased more strongly than their glycosylated counterparts.     

Hierarchical domain‐motion analysis of conformational changes in sarcoplasmic reticulum Ca2+‐ATPase

Sarco(endo)plasmic reticulum Ca²⁺‐ATPase transports two Ca²⁺ per ATP‐hydrolyzed across biological membranes against a large concentration gradient by undergoing large conformational changes. Structural studies with X‐ray crystallography revealed functional roles of coupled motions between the cytoplasmic domains and the transmembrane helices in individual reaction steps. Here, we employed “Motion Tree (MT),” a tree diagram that describes a conformational change between two structures, and applied it to representative Ca²⁺‐ATPase structures. MT provides information of coupled rigid‐body motions of the ATPase in individual reaction steps. Fourteen rigid structural units, “common rigid domains (CRDs)” are identified from seven MTs throughout the whole enzymatic reaction cycle. CRDs likely act as not only the structural units, but also the functional units. Some of the functional importance has been newly revealed by the analysis. Stability of each CRD is examined on the morphing trajectories that cover seven conformational transitions. We confirmed that the large conformational changes are realized by the motions only in the flexible regions that connect CRDs. The Ca²⁺‐ATPase efficiently utilizes its intrinsic flexibility and rigidity to response different switches like ligand binding/dissociation or ATP hydrolysis. The analysis detects functional motions without extensive biological knowledge of experts, suggesting its general applicability to domain movements in other membrane proteins to deepen the understanding of protein structure and function. Proteins 2015; 83:746–756. © 2015 Wiley Periodicals, Inc.     

Rational design of a fusion partner for membrane protein expression in E. coli

We have designed a novel protein fusion partner (P8CBD) to utilize the co‐translational SRP pathway in order to target heterologous proteins to the E. coli inner membrane. SRP‐dependence was demonstrated by analyzing the membrane translocation of P8CBD‐PhoA fusion proteins in wt and SRP‐ffh77 mutant cells. We also demonstrate that the P8CBD N‐terminal fusion partner promotes over‐expression of a Thermotoga maritima polytopic membrane protein by replacement of the native signal anchor sequence. Furthermore, the yeast mitochondrial inner membrane protein Oxa1p was expressed as a P8CBD fusion and shown to function within the E. coli inner membrane. In this example, the mitochondrial targeting peptide was replaced by P8CBD. Several practical features were incorporated into the P8CBD expression system to aid in protein detection, purification, and optional in vitro processing by enterokinase. The basis of membrane protein over‐expression toxicity is discussed and solutions to this problem are presented. We anticipate that this optimized expression system will aid in the isolation and study of various recombinant forms of membrane‐associated protein.     

The crystal structure of ribonuclease A in complex with thymidine‐3′‐monophosphate provides further insight into ligand binding

Thymidine‐3′‐monophosphate (3′‐TMP) is a competitive inhibitor analogue of the 3′‐CMP and 3′‐UMP natural product inhibitors of bovine pancreatic ribonuclease A (RNase A). Isothermal titration calorimetry experiments show that 3′‐TMP binds the enzyme with a dissociation constant (K_d) of 15 μM making it one of the strongest binding members of the five natural bases found in nucleic acids (A, C, G, T, and U). To further investigate the molecular properties of this potent natural affinity, we have determined the crystal structure of bovine pancreatic RNase A in complex with 3′‐TMP at 1.55 Å resolution and we have performed NMR binding experiments with 3′‐CMP and 3′‐TMP. Our results show that binding of 3′‐TMP is very similar to other natural and non‐natural pyrimidine ligands, demonstrating that single nucleotide affinity is independent of the presence or absence of a 2′‐hydroxyl on the ribose moiety of pyrimidines and suggesting that the pyrimidine binding subsite of RNase A is not a significant contributor of inhibitor discrimination. Accumulating evidence suggests that very subtle structural, chemical, and potentially motional variations contribute to ligand discrimination in this enzyme. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Integrated solution to purification challenges in the manufacture of a soluble recombinant protein in E. coli

Apolipoprotein A 1 Milano (ApoA‐1M), the protein component of a high‐density lipoprotein (HDL) mimic with promising potential for reduction of atherosclerotic plaque, is produced at large scale by expression in E. coli. Significant difficulty with clearance of host cell proteins (HCPs) was experienced in the original manufacturing process despite a lengthy downstream purification train. Analysis of purified protein solutions and intermediate process samples led to identification of several major HCPs co‐purifying with the product and a bacterial protease potentially causing a specific truncation of ApoA‐1M found in the final product. Deletion of these genes from the original host strain succeeded in substantially reducing the levels of HCPs and the truncated species without adversely affecting the overall fermentation productivity, contributing to a much more efficient and robust new manufacturing process. Biotechnol. Bioeng. 2010; 105: 239–249. © 2009 Wiley Periodicals, Inc.     

β‐Ionone as putative semiochemical suggested by ligand binding on an odorant‐binding protein of Hylamorpha elegans and electroantennographic recordings

Currently, odorant‐binding proteins (OBPs) are considered the first filter for olfactory information for insects and constitute an interesting target for pest control. Thus, an OBP (HeleOBP) from the scarab beetle Hylamorpha elegans (Burmeister) was identified, and ligand‐binding assays based on fluorescence and in silico approaches were performed, followed by a simulated binding assay. Fluorescence binding assays showed slight binding for most of the ligands tested, including host‐plant volatiles. A high binding affinity was obtained for β‐ionone, a scarab beetle‐related compound. However, the binding of its analogue α‐ionone was weaker, although it is still considered good. On the other hand, through a three‐dimensional model of HeleOBP constructed by homology, molecular docking was carried out with 29 related ligands to the beetle. Results expressed as free binding energy and fit quality (FQ) indicated strong interactions of sesquiterpenes and terpenoids (α‐ and β‐ionone) with HeleOBP as well as some aromatic compounds. Residues such as His102, Tyr105 and Tyr113 seemed to participate in the interactions previously mentioned. Both in silico scores supported the experimental affinity for the strongest ligands. Therefore, the activity of α‐ionone, β‐ionone and 2‐phenyl acetaldehyde at antennal level was studied using electroantenography (EAG). Results showed that the three ligands are electrophysiologically active. However, an aliquot of β‐ionone (represented by 3.0 ng) elicited stronger EAG responses in antennae of males than of females. Finally, the role of these ligands as potential semiochemicals for H. elegans is discussed.     

Mechanism of formation of the C‐terminal β‐hairpin of the B3 domain of the immunoglobulin binding protein G from Streptococcus. II. Interplay of local backbone conformational dynamics and long‐range hydrophobic interactions in hairpin formation

Two peptides, corresponding to the turn region of the C‐terminal β‐hairpin of the B3 domain of the immunoglobulin binding protein G from Streptococcus, consisting of residues 51–56 [IG(51–56)] and 50–57 [IG(50–57)], respectively, were studied by circular dichroism and NMR spectroscopy at various temperatures and by differential scanning calorimetry. Our results show that the part of the sequence corresponding to the β‐turn in the native structure (DDATKT) of the B3 domain forms bent conformations similar to those observed in the native protein. The formation of a turn is observed for both peptides in a broad range of temperatures (T = 283–323 K), which confirms the conclusion drawn from our previous studies of longer sequences from the C‐terminal β‐hairpin of the B3 domain of the immunoglobulin binding protein G (16, 14, and 12 residues), that the DDATKT sequence forms a nucleation site for formation of the β‐hairpin structure of peptides corresponding to the C‐terminal part of all the B domains of the immunoglobulin binding protein G. We also show and discuss the role of long‐range hydrophobic interactions as well as local conformational properties of polypeptide chains in the mechanism of formation of the β‐hairpin structure. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Fluorescence of native single-Trp mutants in the lactose permease from Escherichia coli: structural properties and evidence for a substrate-induced conformational change.

Six single-Trp mutants were engineered by individually reintroducing each of the native Trp residues into a functional lactose permease mutant devoid of Trp (Trp-less permease; Menezes ME, Roepe PD, Kaback HR, 1990, Proc Natl Acad Sci USA 87:1638-1642), and fluorescent properties were studied with respect to solvent accessibility, as well as alterations produced by ligand binding. The emission of Trp 33, Trp 78, Trp 171, and Trp 233 is strongly quenched by both acrylamide and iodide, whereas Trp 151 and Trp 10 display a decrease in fluorescence in the presence of acrylamide only and no quenching by iodide. Of the six single-Trp mutants, only Trp 33 exhibits a significant change in fluorescence (ca. 30% enhancement) in the presence of the substrate analog beta,D-galactopyranosyl 1-thio-beta,D-galactopyranoside (TDG). This effect was further characterized by site-directed fluorescent studies with purified single-Cys W33-->C permease labeled with 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid (MIANS). Titration of the change in the fluorescence spectrum reveals a 30% enhancement accompanied with a 5-nm blue shift in the emission maximum, and single exponential behavior with an apparent KD of 71 microM. The effect of substrate binding on the rate of MIANS labeling of single-Cys 33 permease was measured in addition to iodide and acrylamide quenching of the MIANS-labeled protein. Complete blockade of labeling is observed in the presence of TDG, as well as a 30% decrease in accessibility to iodide with no change in acrylamide quenching. Overall, the findings are consistent with the proposal (Wu J, Frillingos S, Kaback HR, 1995a, Biochemistry 34:8257-8263) that ligand binding induces a conformational change at the C-terminus of helix I such that Pro 28 and Pro 31, which are on one face, become more accessible to solvent, whereas Trp 33, which is on the opposite face, becomes less accessible to the aqueous phase. The findings regarding accessibility to collisional quenchers are also consistent with the predicted topology of the six native Trp residues in the permease.     

An interaction-based analysis of calcium-induced conformational changes in Ca2+ sensor proteins.

Calcium sensor proteins translate transient increases in intracellular calcium levels into metabolic or mechanical responses, by undergoing dramatic conformational changes upon Ca2+ binding. A detailed analysis of the calcium binding-induced conformational changes in the representative calcium sensors calmodulin (CaM) and troponin C was performed to obtain insights into the underlying molecular basis for their response to the binding of calcium. Distance difference matrices, analysis of interresidue contacts, comparisons of interhelical angles, and inspection of structures using molecular graphics were used to make unbiased comparisons of the various structures. The calcium-induced conformational changes in these proteins are dominated by reorganization of the packing of the four helices within each domain. Comparison of the closed and open conformations confirms that calcium binding causes opening within each of the EF-hands. A secondary analysis of the conformation of the C-terminal domain of CaM (CaM-C) clearly shows that CaM-C occupies a closed conformation in the absence of calcium that is distinct from the semi-open conformation observed in the C-terminal EF-hand domains of myosin light chains. These studies provide insight into the structural basis for these changes and into the differential response to calcium binding of various members of the EF-hand calcium-binding protein family. Factors contributing to the stability of the Ca2+-loaded open conformation are discussed, including a new hypothesis that critical hydrophobic interactions stabilize the open conformation in Ca2+ sensors, but are absent in "non-sensor" proteins that remain closed upon Ca2+ binding. A role for methionine residues in stabilizing the open conformation is also proposed.     

Chemical synthesis and characterization of wild‐type and biotinylated N‐terminal domain 1–64 of β2‐glycoprotein I

The antiphospholipid syndrome (APS) is a severe autoimmune disease associated with recurrent thrombosis and fetal loss and characterized by the presence of circulating autoantibodies (aAbs) mainly recognizing the N‐terminal domain (DmI) of β2‐glycoprotein I (β2GpI). To possibly block anti‐β2GpI Abs activity, we synthesized the entire DmI comprising residues 1–64 of β2GpI by chemical methods. Oxidative disulfide renaturation of DmI was achieved in the presence of reduced and oxidized glutathione. The folded DmI (N‐DmI) was purified by RP‐HPLC, and its chemical identity and correct disulfide pairing (Cys4‐Cys47 and Cys32‐Cys60) were established by enzymatic peptide mass fingerprint analysis. The results of the conformational characterization, conducted by far‐ and near‐UV CD and fluorescence spectroscopy, provided strong evidence for the native‐like structure of DmI, which is also quite resistant to both Gdn‐HCl and thermal denaturation. However, the thermodynamic stability of N‐DmI at 37°C was remarkably low, in agreement with the unfolding energetics of small proteins. Of note, aAbs failed to bind to plates coated with N‐DmI in direct binding experiments. From ELISA competition experiments with plate‐immobilized β2GpI, a mean IC₅₀ value of 8.8 μM could be estimated for N‐DmI, similar to that of the full‐length protein, IC₅₀(β2GpI) = 6.4 μM, whereas the cysteine‐reduced and carboxamidomethylated DmI, RC‐DmI, failed to bind to anti‐β2GpI Abs. The versatility of chemical synthesis was also exploited to produce an N‐terminally biotin‐(PEG)₂‐derivative of N‐DmI (Biotin‐N‐DmI) to be possibly used as a new tool in APS diagnosis. Strikingly, Biotin‐N‐DmI loaded onto a streptavidin‐coated plate selectively recognized aAbs from APS patients.