Amino Acid Spacer Research Articles

Optimizing Transfection Efficiency of Spermine Polar Head Cholesterol-Based Cationic Lipids with Amino Acid Linker.

In this work, a series of spermine polar head cholesterol-based cationic lipids with various amino acid spacers were synthesized and evaluated as non-viral gene delivery systems. The physicochemical properties of the resulting lipoplexes, formed from these lipids and DOPE, were assessed, including zeta-potential, DNA binding and DNA protection from serum. Transfection efficiency and cytotoxicity were examined under serum-free and 10-40 % serum-containing conditions. The results showed that the physicochemical properties of cationic lipids, both with and without amino acid spacers, were not significantly different. Cationic liposomes composed of lipid Sper-Ahx-Chol, which has a 6-aminohexanoic acid spacer, and DOPE exhibited greater transfection efficiency in HeLa cells compared to Lipofectamine3000, both in the absence and presence of 10-40 % serum. Additionally, lipid Sper-His-Chol with a histidine spacer and Sper-Ahx-Chol showed higher efficiency than Lipofectamine3000 against HEK293T under 40 % serum conditions. These results suggest that the incorporation of amino acids into the cationic lipids can significantly enhance their DNA delivery efficiency. Specifically, certain amino acid modifications improved transfection efficiency while maintaining low cytotoxicity. Our findings highlight the potential of amino acid-tailored cationic lipids as promising vectors for enhanced DNA delivery.

A trafficking motif alters GEVI activity implicating persistent protein interactions at the membrane

Efficient plasma-membrane expression is critical for genetically encoded voltage indicators (GEVIs). To improve the plasma-membrane expression, we introduced multiple combinations of plasma-membrane trafficking motifs at different positions to members of the Bongwoori family of GEVIs. An improvement from 20% to 27% in the ΔF/F/100 mV depolarization of the plasma membrane was observed when a Golgi transport motif was inserted near the N-terminus in conjunction with an endoplasmic reticulum release motif near the C-terminus of the protein. Unfortunately, this variant was also slower. The weighted tau on of the variant (25 ms) was more than double the original construct (11 ms). The weighted tau off was >20 ms compared with 10 ms for the original GEVI. The voltage range of the GEVI was also shifted to more negative potentials. Insertion of spacer amino acids between the fluorescent-protein domain and the endoplasmic reticulum release motif at the C-terminus rescued the speed of both the tau on and tau off while restoring the voltage range and maintaining the improved voltage-dependent optical signal. These results suggest that while trafficking motifs do improve plasma-membrane expression, they may also mediate persistent associations that affect the functioning of the protein.

Effect of Hydrophobicity and Charge Separation on the Antifouling Properties of Surface-Tethered Zwitterionic Peptides.

Zwitterionic peptides emerge as a class of highly effective antifouling material in a wide range of applications such as biosensors, biomedical devices, and implants. We incorporated neutral amino acid spacers with different hydrophobicities, including serine (Ser), glycine (Gly), and leucine (Leu), into zwitterionic peptides with lysine-glutamic acid repeating units and investigated the structure and antifouling performance of the zwitterionic peptide brushes by surface plasma resonance, surface force apparatus (SFA), and all-atomistic molecular dynamics (MD) simulation techniques. Our results demonstrate that the hydrophilicity of neutral spacers alters the structure and antifouling performance of the peptide-modified surface. Hydrophilic Ser-inserted peptides reduced the interaction between the peptide monolayer and protein foulants, while hydrophobic Leu significantly increased the protein adhesion. SFA force measurements show that the presence of more spacers would increase the adhesion between the peptide monolayer and the modeling foulant lysozyme, especially for the hydrophobic spacers. MD simulations reveal that hydrophilic Ser spacers retain the hydrophilicity of the peptide monolayer and improve the antifouling performance, and Gly spacers give rise to more interchain cross-links. Leu spacers result in a more hydrophobic peptide monolayer, which leads to dehydration of the peptide monolayer and reduces the antifouling performances.

Cation-Anion Interactions, Stability, and IR Spectra of Dicationic Amino Acid-Based Ionic Liquids Probed Using Density Functional Theory.

In this work, we have theoretically studied the dicationic ionic liquids (DILs) constructed from geminal methylimidazolium dication with varying amino acid anions and spacers using density functional theory. Amino acid-based DILs form via strong C-H···O hydrogen bonds. These hydrogen bonds have a significant role in stabilizing the DILs. The higher cation-anion interaction energy in the order of covalent bond energy and liquid density of DILs imply higher thermal stability than their mono analogues. The C-H stretching frequencies are above 3100cm-1 in all complexes and form a signature for DILs. Interestingly, aliphatic and aromatic amino acid anions show similar molecular properties. Overall, the DILs formed from amino acids exhibit high stability and large surface tension and are chemically non-toxic; hence, they can replace inorganic DILs.

The metamorphosis of insects and their regulation

The metamorphosis of insects and their regulation

Antimicrobial alumina nanobiostructures of disulfide- and triazole-linked peptides: Synthesis, characterization, membrane interactions and biological activity.

Due to the its physical-chemical properties, alumina nanoparticles have potential applications in several areas, such as nanobiomaterials for medicinal or orthodontic implants, although the introduction of these devices poses a serious risk of microbial infection. One convenient strategy to circumvent this problem is to associate the nanomaterials to antimicrobial peptides with broad-spectrum of activities. In this study we present two novel synthesis approaches to obtain fibrous type alumina nanoparticles covalently bound to antimicrobial peptides. In the first strategy, thiol functionalized alumina nanoparticles were linked via disulfide bond formation to a cysteine residue of an analog of the peptide BP100 containing a four amino acid spacer (Cys-Ala-Ala-Ala). In the second strategy, alumina nanoparticles were functionalized with azide groups and then bound to alkyne-decorated analogs of the peptides BP100 and DD K through a triazole linkage obtained via a copper(I)-catalyzed cycloaddition reaction. The complete physical-chemical characterization of the intermediates and final materials is presented along with in vitro biological assays and membrane interaction studies, which confirmed the activity of the obtained nanobiostructures against both bacteria and fungi. To our knowledge, this is the first report of aluminum nanoparticles covalently bound to triazole-peptides and to a disulfide bound antimicrobial peptide with high potential for biotechnological applications.

Supramolecular Chiral Helical Ribbons of Tetraphenylethylene-Appended Naphthalenediimide Controlled by Solvent and Induced by l- and d-Alanine Spacers.

Naphthalenediimide-tetraphenylethylene conjugates with an alanine spacer (coded as: NDI-(Ala-TPE)2 ) were synthesized to study the influence of the chirality of the amino acid spacer on its self-assemblies. Here we particularly show that NDI-Ala-TPE bearing l-alanine gives left-handed (M-type) helical superstructure, while d-alanine produces right-handed (P-type) helical ribbons in THF:H2 O at 40:60 % v/v ratio. However, particular aggregates were observed at 20:80 % v/v ratio. Circular dichroism was used to characterise the induction of chirality and the handedness of the helical superstructures, and the microstructure of the self-assembled materials was visualised using scanning electron microscopy while DLS analysis confirmed the formation of particular aggregates in solution.

Abstract 1657: Structure-activity relationship studies and biological evaluation of novel maytansinoids, a class of highly selective tubulin inhibitors

Abstract Introduction and objectives: Maytansine and its analogs (DM1 and DM4) are potent microtubule-targeting compounds that inhibit proliferation of cells during mitosis.1 Unfortunately, their narrow therapeutic window prevents a clinical application of these molecules. So far only T-DM1, an antibody-maytansinoid conjugate targeting the HER2 receptor, has been approved for the treatment of resistant breast cancer. Previous work on maytansinoids showed that their potent cytotoxic activity is related to the nature of the substituent at the C3 acyloxy side chain. In order to harness the potential of compounds of the maytansinoid family while diminishing dose-limiting side effects, we synthesized a library of novel analogs which can be attached to serum albumin in vivo through an acid-sensitive linker ensuring release of the active maytansinoid at the tumor site. All novel maytansine analogs contain a keto moiety as an attachment point for the linker and differ in their substitution pattern at C3. The analogs we studied can be classified into those containing an amino acid spacer between C3 and the keto group (maytansine-type) and those obtained through direct esterification at the C3-OH-position with various carboxylic acids (ansamitocin-type). Methods: All the newly synthesized molecules were screened for their in vitro cytotoxicity against 11 cancer cell lines. In order to explore the influence of the maytansine functional groups at C3, we designed novel analogs with different amino acid spacers at this position. We evaluated the influence of the chain length, the degree of steric hindrance, the need of the chiral center and the influence of the N-methyl on the cytotoxicity as well as stability in murine and human blood plasma. Moreover, we studied the structure-activity relationship of the ansamitocin-type of analogs including the influence of the chain length, the degree of steric hindrance and the effect of introducing different heteroatoms in the alkyl chain. Result and conclusion: A total of 32 new maytansinoid analogs were synthesized, and seven of them were found to be more potent than the parent drug maytansine in inhibiting the growth of human cancer cells in vitro. Clear SARs were identified for both classes of compounds. Based on these studies, lead compounds have been selected for creating albumin-binding derivatives and their further in vivo evaluation.2 1) W. C. Widdison et al, J. Med. Chem., 49: 4392-4408 (2006); K. A. Poon et al, Toxicology and Applied Pharmacology, 273: 298-313 (2013); H. L. Perez et al, Drug Discovery Today, 19:869-881 (2014) 2) Abstract "In vivo efficacy of novel acid-sensitive albumin-binding Maytansinoid-based prodrugs in human cancer xenograft models in nude mice" Citation Format: Friederike I. Nollmann, Patricia Perez Galan, Javier Garcia Fernandez, Heidi K. Walter, Johannes P. Magnusson, Federico Medda, Felix Kratz, Stephan D. Koester, Khalid Abu Ajaj, Lara Pes, Serghei Chercheja, Anna Warnecke. Structure-activity relationship studies and biological evaluation of novel maytansinoids, a class of highly selective tubulin inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1657.

Dynamic Modulation of Circularly Polarized Luminescence in Photoresponsive Assemblies

AbstractA photochromic molecule composed of a helical tetrathiazole framework and two pyrene units bridged by amino acid spacers exhibited on‐off photoswitching of circularly polarized luminescence (CPL) of a pyrene‐excimer‐like state in a molecularly dispersed state. The pyrene‐modified chiral tetrathiazole is employed as a building unit of supramolecular aggregates and self‐assembled into spherical nanoparticles in a methylcyclohexane‐based mixed solvent system. The spherical assemblies showed an enhancement of CPL activity compared to that of the molecularly dispersed state. UV light irradiation of the self‐assembled state gave rise to a 6π‐electrocyclization reaction in the photoresponsive tetrathiazole framework. The photoreaction of the self‐assembly component led to efficient emission quenching via energy transfer from the pyrene unit to the colored photochrome center in the aggregates. The CPL activity was diminished along with this emission quenching. The photoreaction also brought about the clouding of the solution due to a secondary aggregation of spherical assemblies. The CPL activity as well as the secondary aggregation were reversibly modulated by successive photoirradiation using UV and visible (>440 nm) light.

Surface enhanced Raman scattering and quantum‐mechanical calculations on self‐assembling oligopeptides

AbstractThe surface‐enhanced Raman scattering spectra of 5 amphiphilic oligopeptides derived from EAK16 (AEAEAKAK)2, used as biomimetic coatings for medical devices, were obtained at 10−5–10−6 M in order to study the effects of systematic amino acid substitution along the peptide chain on the corresponding interaction with Ag colloidal nanoparticles. In addition, quantum‐mechanical data on 2 of the examined peptides were very useful for clarifying the assignment of bands, widely debated in the literature. In general, the peptide–nanoparticles interaction takes place through the COO− groups. The substitution of Ala by 2‐aminobutanoic acid in the sequence, corresponding to an increase of the hydrophobic chain length, is able to affect the peptide–Ag colloid interaction. It strengthens the interaction with NH3+ groups, mediated by the Cl− anions present in the colloidal solution, although the charge transfer interaction with the COO− ions remains the dominant interaction mechanism. When Tyr substitutes the hydrophobic Ala, the interaction mechanism is strongly affected because it takes mainly place through the Tyr residues, where the aromatic rings are predominantly perpendicular to the silver surface, partly as tyrosinate ion. Thus, the addition of the Arg‐Gly‐Asp sequence, useful to provide control on the bioactivity of the bone regeneration materials, does not change the interactions with the colloid, because the spacer amino acid substitution is the main factor affecting the peptide–nanoparticle interactions.

Triple Vectors Expand AAV Transfer Capacity in the Retina.

Retinal gene transfer with adeno-associated viral (AAV) vectors holds great promise for the treatment of inherited retinal degenerations (IRDs). One limit of AAV is its transfer capacity of about 5 kb, which can be expanded to about 9 kb, using dual AAV vectors. This strategy would still not suffice for treatment of IRDs such as Usher syndrome type 1D or Alström syndrome type I (ALMS) due to mutations in CDH23 or ALMS1, respectively. To overcome this limitation, we generated triple AAV vectors, with a maximal transfer capacity of about 14 kb. Transcriptomic analysis following triple AAV transduction showed the expected full-length products along a number of aberrant transcripts. However, only the full-length transcripts are efficiently translated in vivo. We additionally showed that approximately 4% of mouse photoreceptors are transduced by triple AAV vectors and showed correct localization of recombinant ALMS1. The low-photoreceptor transduction levels might justify the modest and transient improvement we observe in the retina of a mouse model of ALMS. However, the levels of transduction mediated by triple AAV vectors in pig retina reached 40% of those observed with single vectors, and this bodes well for further improving the efficiency of triple AAV vectors in the retina.

(R-X-R)4 -Motif Peptides Containing Conformationally Constrained Cyclohexane-Derived Spacers: Effect on Cellular Uptake.

Arginine-rich peptides having the (R-X-R)n motif are among the most effective cell-penetrating peptides (CPPs). Herein we report a several-fold increase in the efficacy of such CPPs if the linear flexible spacer (-X-) in the (R-X-R) motif is replaced by constrained cyclic 1,4-substituted-cyclohexane-derived spacers. Internalization of these oligomers in mammalian cell lines was found to be an energy-dependent process. Incorporation of these constrained, non-proteinogenic amino acid spacers in the CPPs is shown to enhance their proteolytic stability.

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Abstract 4017: Development and application of an immunohistochemistry-based assay for evaluating functional and accessible folate receptor expression in vivo

Abstract With the development of molecularly-targeted therapeutics, it is critical to have a reliable method for predicting response to therapy of patients with target-expressing diseases. Folate receptor (FR) is overexpressed in a number of disease states while it is low in most healthy individuals with the exception of the kidney. FR-targeted small molecule drug conjugates (SMDCs) are currently under development and have shown promising pre-clinical and clinical results. Two premises that a patient would respond to the folate-drug conjugates are: 1). FR protein expressed in diseased tissues is functionally competent for folate binding and 2). functional FR is accessible to intravenously-infused SMDCs. Although anti-FR antibody-based immunohistochemical (IHC) assays are being developed as companion diagnostic strategies for antibody-drug conjugate (ADC) therapies, it fails to address the functionality and accessibility of FR in an in vivo context. To circumvent these limitations, we have developed an FR-targeted, crosslinkable small molecule reporter conjugate (SMRC), FRRC, which contains three modules: 1. a high-affinity folate ligand that binds with FR on diseased cells; 2. a small hapten, FITC, which can be detected by anti-FITC antibodies; and 3. an amino acid spacer in between these two modules that can crosslink FRRC to FR in-situ during formalin fixation. After intravenous (i.v.) injection, FRRC would dock to the accessible and functional receptors in vivo. By processing biopsied tissues and performing anti-FITC IHC staining, we can evaluate the cellular localization and relative abundance of functional FR in heterogeneous tissues. To evaluate our design, we synthesized a compound we refer to as FRRC and examined its properties both in vitro and in vivo. By testing FRRC in cell lines, we found that it can bind to FR and is detected in formalin-fixed cells by anti-FITC immunostaining. To validate this assay in vivo, FRRC was injected via the tail vein into mice bearing FR-positive KB tumor xenografts and its specific accumulation and kinetics in tumor and other tissues were evaluated by performing anti-FITC IHC staining on formalin-fixed paraffin embedded tissues. Our results were shown to be in agreement with previous folate-based functional FR imaging and bio-distribution studies. In contrast, EC17, which lacks the crosslinkable spacer module, showed significantly reduced binding in in vitro and in vivo assays. Owing to its modular design, additional SMRCs with different ligands and small haptens (rhodamine, DNP) have also been designed to assess the functional binding of other receptor and membrane-expressed proteins. In conclusion, our assay is an effective tool for evaluating functional and accessible receptor expression in vitro and in vivo, and has the potential to be useful in patient or disease selection for our SMDC therapeutics. Citation Format: Haiyan Chu, Jonathan M. Shillingford, Nikki Parker, Melissa Nelson, Jeremy F. Vaughn, Albert Felten, Yingjuan Lu, Joseph A. Reddy, Iontcho R. Vlahov, Christopher P. Leamon. Development and application of an immunohistochemistry-based assay for evaluating functional and accessible folate receptor expression in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4017. doi:10.1158/1538-7445.AM2017-4017

Synthesis of pH-Sensitive Fluorescein Grafted Cellulose Nanocrystals with an Amino Acid Spacer

Fluorescent labeled nanoparticles are expected to have potential applications in biological and imaging systems. In this study, in order to construct the fluorescent nanoparticles for pH-sensing, l-leucine amino acid is used as a spacer linker between cellulose nanocrystals (CNCs) and a pH-indicator dye (5 (and 6)-carboxy-2′,7′-dichlorofluorescein, CDCF). Characterization by Fourier transform infrared (FTIR), nuclear magnetic resonance (NMR), and X-ray photoelectron spectroscopy (XPS) is adopted to follow the chemical modification, whereas the morphology is determined by atomic force microscopy (AFM). The crystallinity determined by NMR for the CNCs, A-CNCs, and F-A-CNCs is 78.32, 79.84, and 79.52, respectively. The quantitative analysis by elemental analysis revealed that the degree of cellulose hydroxyl group substitution (DS) of A-CNCs and F-A-CNCs is 0.05 and 0.27, respectively. According to the obtained results, CDCF fluorescein could be successfully labeled onto CNCs via l-leucine amino acid spacer linking, and the cellulose structure is stable during the labeling reaction. The fluorescent properties of the yielding pH-sensitive fluorescent CNCs (F-A-CNCs) are characterized by fluorescence spectrometer and imaged by confocal laser scanning microscopy (CLSM). The result shows that the fluorescence intensity of F-A-CNCs increases with increasing pH of the buffer from 2.28 to 10.84.

Conopeptide-Derived κ-Opioid Agonists (Conorphins): Potent, Selective, and Metabolic Stable Dynorphin A Mimetics with Antinociceptive Properties

Opioid receptor screening of a conopeptide library led to a novel selective κ-opioid agonist peptide (conorphin T). Intensive medicinal chemistry, guided by potency, selectivity, and stability assays generated a pharmacophore model supporting rational design of highly potent and selective κ-opioid receptor (KOR) agonists (conorphins) with exceptional plasma stability. Conorphins are defined by a hydrophobic benzoprolyl moiety, a double arginine sequence, a spacer amino acid followed by a hydrophobic residue and a C-terminal vicinal disulfide moiety. The pharmacophore model was supported by computational docking studies, revealing receptor-ligand interactions similar to KOR agonist dynorphin A (1-8). A conorphin agonist inhibited colonic nociceptors in a mouse tissue model of chronic visceral hypersensitivity, suggesting the potential of KOR agonists for the treatment of chronic abdominal pain. This new conorphine KOR agonist class and pharmacophore model provide opportunities for future rational drug development and probes for exploring the role of the κ-opioid receptor.

Synthesis and in vitro Evaluation of Polymeric Prodrug of Ibuprofen with Amino Acid Spacer.

The present work is an agreement with simple and efficient method of improving the therapeutic efficacy of ibuprofen by masking its acidic moiety. It aims to reduce gastrointestinal side effects by controlling the rate, duration and site of release. This is achieved by synthesis and evaluation of polymeric prodrug of ibuprofen with natural polymer sodium alginate. The synthesis was supported by N-protected serine as spacer due to chemical incompatibility of drug and polymer. Synthesized prodrug was characterized for confirmation of said structures. The in-vitro dissolution profile of ibuprofen-alginate prodrug showed that the release of the drug is significantly higher in case of pH 7.2 buffer as compared to ibuprofen, which might be due to ester group adjacent to drug get hydrolyzed. The hydrolysis was found to be with faster rate in alkaline media than that of in acidic media.

Stereochemistry of amino acid spacers determines the pharmacokinetics of (111)In-DOTA-minigastrin analogues for targeting the CCK2/gastrin receptor.

The metabolic instability and high kidney retention of minigastrin (MG) analogues hamper their suitability for use in peptide-receptor radionuclide therapy of CCK2/gastrin receptor-expressing tumors. High kidney retention has been related to N-terminal glutamic acids and can be substantially reduced by coinjection of polyglutamic acids or gelofusine. The aim of the present study was to investigate the influence of the stereochemistry of the N-terminal amino acid spacer on the enzymatic stability and pharmacokinetics of (111)In-DOTA-(d-Glu)6-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 ((111)In-PP11-D) and (111)In-DOTA-(l-Glu)6-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 ((111)In-PP11-L). Using circular dichroism measurements, we demonstrate the important role of secondary structure on the pharmacokinetics of the two MG analogues. The higher in vitro serum stability together with the improved tumor-to-kidney ratio of the (d-Glu)6 congener indicates that this MG analogue might be a good candidate for further clinical study.

Advanced strategies for efficient macrocyclic Cu(I)-catalyzed cycloaddition of azides.

An advanced strategy for efficient macrocyclic Cu(I)-catalyzed cycloaddition is described. The key features include employing azide-iodoalkyne cycloadditions (CuAiAC), low catalyst loadings, relatively high concentrations (30 mM → 300 mM), and application to continuous flow. The remarkably efficient new tool affords a variety of macrocyclic skeletons having either different alkyl, aryl, or amino acid spacers in high yields (70-97%). The macrocyclic CuAiAC process affords macrocycles having an iodotriazole moiety that can be further functionalized using standard Pd-catalyzed cross-couplings.

A Mechanistic Investigation of the C-Terminal Redox Motif of Thioredoxin Reductase from Plasmodium falciparum

High-molecular mass thioredoxin reductases (TRs) are pyridine nucleotide disulfide oxidoreductases that catalyze the reduction of the disulfide bond of thioredoxin (Trx). Trx is responsible for reducing multiple protein disulfide targets in the cell. TRs utilize reduced β-nicotinamide adenine dinucleotide phosphate to reduce a bound flavin prosthetic group, which in turn reduces an N-terminal redox center that has the conserved sequence CICVNVGCCT, where CIC is denoted as the interchange thiol while the thiol involved in charge-transfer complexation is denoted as CCT. The reduced N-terminal redox center reduces a C-terminal redox center on the opposite subunit of the head-to-tail homodimer, the C-terminal redox center that catalyzes the reduction of the Trx-disulfide. Variations in the amino acid sequence of the C-terminal redox center differentiate high-molecular mass TRs into different types. Type Ia TRs have tetrapeptide C-terminal redox centers of with a GCUG sequence, where U is the rare amino acid selenocysteine (Sec), while the tetrapeptide sequence in type Ib TRs has its Sec residue replaced with a conventional cysteine (Cys) residue and can use small polar amino acids such as serine and threonine in place of the flanking glycine residues. The TR from Plasmodium falciparum (PfTR) is similar in structure and mechanism to type Ia and type Ib TRs except that the C-terminal redox center is different in its amino acid sequence. The C-terminal redox center of PfTR has the sequence G534CGGGKCG541, and we classify it as a type II high-molecular mass TR. The oxidized type II redox motif will form a 20-membered disulfide ring, whereas the absence of spacer amino acids in the type I motif results in the formation of a rare eight-membered ring. We used site-directed mutagenesis and protein semisynthesis to investigate features of the distinctive type II C-terminal redox motif that help it perform catalysis. Deletion of Gly541 reduces thioredoxin reductase activity by ∼50-fold, most likely because of disruption of an important hydrogen bond between the amide NH group of Gly541 and the carbonyl of Gly534 that helps to stabilize the β–turn−β motif. Alterations of the 20-membered disulfide ring either by amino acid deletion or by substitution resulted in impaired catalytic activity. Subtle changes in the ring structure and size caused by using semisynthesis to substitute homocysteine for cysteine also caused significant reductions in catalytic activity, demonstrating the importance of the disulfide ring’s geometry in making the C-terminal redox center reactive for thiol–disulfide exchange. The data suggested to us that the transfer of electrons from the N-terminal redox center to the C-terminal redox center may be rate-limiting. We propose that the transfer of electrons from the N-terminal redox center in PfTR to the type II C-terminal disulfide is accelerated by the use of an “electrophilic activation” mechanism. In this mechanism, the type II C-terminal disulfide is polarized, making the sulfur atom of Cys540 electron deficient, highly electrophilic, and activated for thiol–disulfide exchange with the N-terminal redox center. This hypothesis was investigated by constructing chimeric PfTR mutant enzymes containing C-terminal type I sequences GCCG and GCUG, respectively. The PfTR-GCCG chimera had 500-fold less thioredoxin reductase activity than the native enzyme but still reduced selenocystine and lipoic acid efficiently. The PfTR-GCUG chimera had higher catalytic activity than the native enzyme with Trx, selenocystine, and lipoic acid as substrates. The results suggested to us that (i) Sec in the mutant enzyme accelerated the rate of thiol–disulfide exchange between the N- and C-terminal redox centers, (ii) the type II redox center evolved for efficient catalysis utilizing Cys instead of Sec, and (iii) the type II redox center of PfTR is partly responsible for substrate recognition of the cognate PfTrx substrate relative to noncognate thioredoxins.