Side-chain Amino Research Articles

Effective Quenchers Are Required to Eliminate the Interference of Substrate: Cofactor Binding in the HAT Scintillation Proximity Assay.

Histone acetyltransferases (HATs) mediate the transfer of an acetyl group from the cofactor, acetyl-CoA, to the side chain amino group of specific lysines in diverse protein substrates, most notably nuclear histones. The deregulation of HATs is connected to a number of disease states. Reliable and rapid biochemical assays for HATs are critical for understanding biological functions of protein acetylation, as well as for screening small-molecule inhibitors of HAT enzymes. In this report, we present a scintillation proximity assay (SPA) for the measurement of HAT enzymatic activities. The acetyl donor was [(3)H]Ac-CoA, and a biotin-modified histone peptide served as the HAT substrate. After the HAT reaction, streptavidin-coated beads were added to induce proximity of acetylated substrate to the scintillant molecules. However, we observed strong nonspecific binding between the cofactor and the histone peptide substrates, which adversely complicated the SPA performance. To prevent this problem, a set of chemical agents were evaluated to eliminate the cofactor-substrate interaction, thus providing reliable SPA readings. With optimization, the SPA showed consistent and robust performance for HAT activity measurement and HAT inhibitor evaluation. Overall, this mix-and-measure assay does not require any washing procedure, can be utilized in the microplate format, and is well suited for high-throughput screening of HAT chemical modulators.

Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia

The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P(1),P(5)-di(adenosine-5') pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5'-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5'-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl(-) channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia.

Exploring the post-polymerization modification of side-chain amino acid containing polymers via Michael addition reactions

An efficacious protocol for the aza-Michael addition of a C-terminus modified phenylalanine side-chain containing polymer (Michael donor) was demonstrated with various Michael acceptors. The aza-Michael addition reactions were carried out at 50°C in anhydrous methanol, which is a protic solvent, to enhance the advancement of the reactions. 1H and inverse gated 13C NMR spectroscopy were utilized to qualitatively deliver comprehensive data on the reaction progress. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) provided quantitative information about the modification of the polymer. The extent of change in the aza-Michael addition varied with different Michael acceptors and decreased in the order of acrylate>acrylamide>methacrylate. The present study opens up a library of polymers with functional modifications in the side-chain.

Vanadium(V) complex with Schiff-base ligand containing a flexible amino side chain: Synthesis, structure and reactivity

The Schiff-base ligand (H2salhyhNH3)Cl (1) derived from salicylaldehyde and 6-aminohexanoic acid hydrazide hydrochloride reacts with ammonium metavanadate in methanol solution to yield the dioxidovanadium(V) complex [VO2(salhyhNH3)] (2). The utilized hydrazone ligand contains a flexible and protonated amino side chain. Crystallization from methanol affords complex 2 in the monoclinic space group P21/n, whereas crystallization from a methanol/water mixture 1:1 yields crystals, containing a water molecule of crystallization per two formula units (2⋅1/2H2O), in the orthorhombic space group Pbcn. In both cases the protonated amino group compensates the negative charge on the dioxidovanadium moiety and is involved in an extensive hydrogen bonding network particularly including the oxido groups from neighboring vanadium complexes. The reactivity of complex 2 toward protonation in aqueous solution has been investigated by spectrophotometric titrations and is characterized by two subsequent protonation steps at the hydrazide nitrogen atom of the ligand system and an oxido group leading to the formation of an oxidohydroxidovanadium(V) species with corresponding pKa values of 3.2 and 2.9, respectively. With larger excess of acid the oxidohydroxidovanadium(V) species starts to form the corresponding anhydride. The formation of the anhydride is strongly favored in the presence of methanol. The reaction of complex 2 with hydrogen peroxide in methanol solution leads to the formation of an oxidoperoxidovanadium(V) species, whereas in aqueous solution the addition of one equivalent of acid is required. Complex 2 catalyzes the oxidation of methylphenylsulfane to the corresponding sulfoxide in methanol/dichloromethane mixture using hydrogen peroxide as oxidant at room temperature.

Chloroquine and chloroquinoline derivatives as models for the design of modulators of amyloid Peptide precursor metabolism.

The amyloid precursor protein (APP) plays a central role in Alzheimer's disease (AD). Preventing deregulated APP processing by inhibiting amyloidogenic processing of carboxy-terminal fragments (APP-CTFs), and reducing the toxic effect of amyloid beta (Aβ) peptides remain an effective therapeutic strategy. We report the design of piperazine-containing compounds derived from chloroquine structure and evaluation of their effects on APP metabolism and ability to modulate the processing of APP-CTF and the production of Aβ peptide. Compounds which retained alkaline properties and high affinity for acidic cell compartments were the most effective. The present study demonstrates that (1) the amino side chain of chloroquine can be efficiently substituted by a bis(alkylamino)piperazine chain, (2) the quinoline nucleus can be replaced by a benzyl or a benzimidazole moiety, and (3) pharmacomodulation of the chemical structure allows the redirection of APP metabolism toward a decrease of Aβ peptide release, and increased stability of APP-CTFs and amyloid intracellular fragment. Moreover, the benzimidazole compound 29 increases APP-CTFs in vivo and shows promising activity by the oral route. Together, this family of compounds retains a lysosomotropic activity which inhibits lysosome-related Aβ production, and is likely to be beneficial for therapeutic applications in AD.

SCMPSP: Prediction and characterization of photosynthetic proteins based on a scoring card method.

BackgroundPhotosynthetic proteins (PSPs) greatly differ in their structure and function as they are involved in numerous subprocesses that take place inside an organelle called a chloroplast. Few studies predict PSPs from sequences due to their high variety of sequences and structues. This work aims to predict and characterize PSPs by establishing the datasets of PSP and non-PSP sequences and developing prediction methods.ResultsA novel bioinformatics method of predicting and characterizing PSPs based on scoring card method (SCMPSP) was used. First, a dataset consisting of 649 PSPs was established by using a Gene Ontology term GO:0015979 and 649 non-PSPs from the SwissProt database with sequence identity <= 25%.- Several prediction methods are presented based on support vector machine (SVM), decision tree J48, Bayes, BLAST, and SCM. The SVM method using dipeptide features-performed well and yielded - a test accuracy of 72.31%. The SCMPSP method uses the estimated propensity scores of 400 dipeptides - as PSPs and has a test accuracy of 71.54%, which is comparable to that of the SVM method. The derived propensity scores of 20 amino acids were further used to identify informative physicochemical properties for characterizing PSPs. The analytical results reveal the following four characteristics of PSPs: 1) PSPs favour hydrophobic side chain amino acids; 2) PSPs are composed of the amino acids prone to form helices in membrane environments; 3) PSPs have low interaction with water; and 4) PSPs prefer to be composed of the amino acids of electron-reactive side chains.ConclusionsThe SCMPSP method not only estimates the propensity of a sequence to be PSPs, it also discovers characteristics that further improve understanding of PSPs. The SCMPSP source code and the datasets used in this study are available at http://iclab.life.nctu.edu.tw/SCMPSP/.

RAFT synthesis of polyethylene glycol (PEG) and amino-functionalized amphiphilic copolymers for dispersing carbon nanofibers

Quaternary copolymer dispersants containing polyethylene glycol (PEG) and amino side chains were synthesized via RAFT and were used to non-covalently functionalize CNFs.

Synthesis of amino acid based covalently cross-linked polymeric gels using tetrakis(hydroxymethyl) phosphonium chloride as a cross-linker

This work reports the design and synthesis of side-chain amino acid containing cross-linked polymer gels with high mechanical strength. The Boc-leucine methacryloyloxyethyl ester (Boc-Leu-HEMA) has been polymerized by reversible addition-fragmentation chain transfer (RAFT) polymerization to obtain the corresponding polymer, P(Boc-Leu-HEMA). After Boc group deprotection under acidic condition and pH neutralization amino groups remain in the polymer side chain, which were reacted with tetrakis(hydroxymethyl) phosphonium chloride (THPC) to obtain chemically cross-linked gels. The mechanical properties of gels have been studied by rheological measurements, which reveal that mechanical strength increases as weight% of polymer in solution, cross-linking density and polarity of solvents increases, and decreases as molecular weight of polymers increases. A plateau of storage modulus (G′) over a wide range of angular frequency demonstrates that viscoelastic property of gels is independent of angular frequency. Field emission scanning electron microscopy (FE-SEM) studies of cross-sectioned gels validate rheological observation that as the G′ values of gel increases, porosity of gel decreases.

Anticancer activity and DNA binding property of the dimers of triphenylethylene–coumarin hybrid with two amino side chains

Novel dimers of triphenylethylene–coumarin hybrid containing two amino side chains were designed and synthesized by the condensation of three dicarboxylic acids with the amino monomeric hybrids catalyzed by HATU and DIPEA at room temperature. All the dimeric compounds exhibited significant anti-proliferative activity against four cancer cells at IC50 of near 10 μM, and higher than their corresponding monomeric compounds except compound 9b. Among them, compounds 9a (R = NEt2) linked by sebacamide and 8b (R = piperidinyl) linked by adipamide were the best ones, respectively. UV–Vis, fluorescence, and circular dichroism (CD) spectroscopies and thermal denaturation exhibited that the monomeric compounds 6b and the dimers 7b, 8b, and 9b had significant interactions with Ct-DNA by the intercalative mode of binding. The order of their anti-proliferative activities was 8b > 7b> 6b > 9b, in accordance with that of their DNA binding properties. Novel dimers of triphenylethylene–coumarin hybrid containing two amino side chains were designed and synthesized. All the dimeric compounds exhibited significant anti-proliferative activity against cancer cells. Both the length of the linker and the basic amino group had certain effects on their anti-proliferative activity. Anticancer activity and DNA binding property of the dimers of triphenylethylene–coumarin hybrid with two amino side chains

Specific counterion repercussions on the thermal, pH-response, and electrochemical properties of side-chain leucine based chiral polyelectrolytes.

Effects of counterions of side chain amino acid based polyelectrolytes (PEs) on the solubility in aqueous medium, pH responsiveness, thermal properties, and ionic conductivities have been appraised. Deprotection of the tert-butyl carbamate (Boc) group from poly(Boc-l-leucine methacryloyloxyethyl ester) [P(Boc-l-Leu-HEMA)] was carried out to produce PE with trifluoroacetate as an associative counteranion (1a). PEs with bis(trifluoromethylsulfonyl)imide and hexafluorophosphate counteranion were prepared through anion exchange reactions of 1a. Protonation of the neutralized polymer (2) obtained from 1a, followed by anion exchange, leads to the production of miscellaneous PEs bearing different counteranions, such as tetrafluoroborate, trifluoromethanesulfonate, chloride, and nitrate. Differential scanning calorimetry traces of the PEs reveal that the comparatively larger and weakly coordinated counteranions require less thermal energy to dissociate, and thus, the glass transition temperature (Tg) of the PEs fall off with an increase in the size of the counteranion. A remarkable conductivity of 2.1 mS/cm was obtained in deionized water when Cl(-) acted as the counteranion. Steric and electronic factors of the counteranion induce a change of transition pH in different PEs, although the chiroptical nature was retained, as confirmed by circular dichroism spectroscopy.

Photostability of cosmetic UV filters on mammalian skin under UV exposure.

Previous studies showed that the common UV filter substances benzophenone-3 (BP-3), butyl methoxydibenzoylmethane (BM-DBM), octocrylene (OCR), ethylhexyl methoxycinnamate (EHMC), ethylhexyl salicylate (EHS) and ethylhexyl triazone (EHT) were able to react with amino side chains of different proteins in vitro. To transfer the results to mammalian skin conditions, sunscreen products were applied on both prepared fresh porcine skin and glass plates, followed by UV irradiation and the determination of depletion of the respective UV filters. Significantly lower recoveries of the UV filters extracted from skin samples than from glass plates indicated the additional reaction of the UV filters with skin constituents, when proteins will be the most important reactants. Among the products tested, BP-3 showed the greatest differences in recoveries between glass and skin samples of about 13% and 24% after 2 and 4 h of irradiation, respectively, followed by EHS > BM-DBM > OCR > EHMC > EHT. The obtained results raise the question, whether the common in vitro evaluations of sunscreens, using inert substrate materials like roughened quartz or polymethyl methacrylate (PMMA) plates are really suitable to fully replace in vivo methods, as they cannot include skin-typical reactions.

Investigation of the Mechanism of Racemization of Litronesib in Aqueous Solution: Unexpected Base‐Catalyzed Inversion of a Fully Substituted Carbon Chiral Center

Mitosis inhibitor (R)-litronesib (LY2523355) is a 1,3,4-thiadiazoline-bearing phenyl and N-(2-ethylamino)ethanesulfonamido-methyl substituents on tetrahedral C5. Chiral instability has been observed at pH 6 and above with the rate of racemization increasing with pH. A positively charged trigonal intermediate is inferred from the fact that p-methoxy substituent on the phenyl accelerated racemization, whereas a p-trifluoromethyl substituent had the opposite effect. Racemization is proposed to occur through a relay mechanism involving intramolecular deprotonation of the sulfonamide by the side chain amino group and attack of the sulfonamide anion on C5, cleaving the C5S bond, to form an aziridine; heterolytic dissociation of the aziridine yields an ylide. This pathway is supported by (1) a crystal structure providing evidence for a hydrogen bond between the sulfonamide NH and the amino group, (2) effects of substituents on the rate of racemization, and (3) computational studies. This racemization mechanism results from neighboring group effects in this densely functionalized molecule. Of particular novelty is the involvement of the side-chain secondary amino group, which overcomes the weak acidity of the sulfonamide by anchimeric assistance.

Molecular modeling and analysis of human and plant endo-β-N-acetyl- glucosaminidases for mutations effects on function.

Endo- β-N-acetylgucosaminidases (ENGases) are the enzymes that catalyze both hydrolysis and transglycosylation reactions. It is of interest to study ENGases because of their ability to synthesize glycopeptides. Homology models of Human, Arabidopsis thaliana and Sorghum ENGases were developed and their active sites marked based on information available from Arthrobacter protophormiae (PDB ID: 3FHQ) ENGase. Further, these models were docked with the natural substrate GlcNAc-Asn and the inhibitor Man3GlcNAc-thiazoline. The catalytic triad of Asn, Glu and Tyr (N171, E173 and Y205 of bacteria) were found to be conserved across the phyla. The crucial Y299F mutation showing 3 times higher transglycosylation activity than in wild type Endo-A is known. The hydrolytic activity remained unchanged in bacteria, while the transglycosylation activity increased. This Y to F change is found to be naturally evolved and should be attributing higher transglycosylation rates in human and Arabidopsis thaliana ENGases. Ligand interactions Ligplots revealed the interaction of amino acids with hydrophobic side chains and polar uncharged side chain amino acids. Thus, structure based molecular model-ligand interactions provide insights into the catalytic mechanism of ENGases and assist in the rational engineering of ENGases.

Catalysis of ubiquitin transfer by the RBR ubiquitin ligase parkin

Mutations in the Parkin gene are responsible for an autosomal recessive form of Parkinson's disease. The parkin protein is a RING1-In-Between-RING2 (RBR) E3 ubiquitin ligase, which functions through a two-step mechanism involving a parkin~ubiquitin thioester intermediate [1]. However, compared to other ubiquitin ligases, parkin exhibits low basal activity and requires activation both in vitro and in cells. As parkin is neuroprotective in various models of Parkinson's disease, understanding how it catalyses ubiquitin transfer will be critical. We previously reported the crystal structure of full-length parkin [2]. The structure shows parkin in an auto-inhibited state and provides insight into how it is activated. The RING0 domain occludes the ubiquitin acceptor site Cys431 in RING2 whereas a novel Repressor Element of Parkin (REP) binds RING1 and blocks its E2-binding site. Remarkably, mutations that disrupt these inhibitory interactions activate parkin both in vitro and in cells. The structure also reveals that His433 and Glu444 form a catalytic dyad adjacent to Cys431. Here, we show that His433 catalyses the acyl transfer of ubiquitin carboxy terminus from Cys431 to a target lysine side-chain amino group. Mutation of His433 does not affect UbcH7~ubiquitin discharging or thioester intermediate formation, but prevents formation of polyubiquitin chains on parkin. However, mutation of His433 does not affect significantly parkin's mitochondrial recruitment and substrate ubiquitination, suggesting that other factors might be at play in vivo. We also investigate the catalytic role of other residues located around the Cys431, such as Trp462. The work provides insight into the mechanism of ubiquitination by RBR E3 ligases with important implications for Parkinson's disease.

Microwave-assisted synthesis of cyclic phosphopeptide on solid support.

Phosphopeptides are important tools for studying intracellular signal transduction events in vitro and in vivo and are also potential drugs due to their direct competition with phosphoprotein recognition elements. Cyclization has been demonstrated to improve peptide selectivity, metabolic stability, and bioavailability. However, cyclic phosphopeptide synthesis may not be straightforward due to the sterically hindered phosphorylated side-chain amino acid derivatives. One option to overcome this hurdle is to use microwave-assisted synthesis, which has been shown to increase efficiency and reduce synthesis time. Herein, a detailed protocol is provided for synthesizing cyclic phosphopeptides using automated microwave. The overall synthesis duration was reduced and yields increased compared with a manual conventional method. This method provides a general, fast and facile way to synthesize cyclic peptides, demonstrating the synthesis of cyclic phosphorylated peptides which are known to be among the most challenging to produce.

Facile RAFT synthesis of side-chain amino acids containing pH-responsive hyperbranched and star architectures

This work reports the design and synthesis of amino acid-based hyperbranched polymers via the combination of self-condensing vinyl polymerization (SCVP) and reversible addition–fragmentation chain transfer (RAFT) polymerization from tert-butyl carbamate (Boc)-L-valine acryloyloxyethyl ester (Boc-Val-HEA) and S-(4-vinyl)benzyl S′-butyltrithiocarbonate (VBBT) with variable degrees of branching (DB), molecular weights (Mn), and chain end functionalities. Copolymerization kinetics reveal that the molecular weight increases and the DB decreases linearly with time as the branch length increases with the conversion of the Boc-Val-HEA monomer. These hyperbranched polymers, P(Boc-Val-HEA-co-VBBT), with tuneable Mn and DB have been further employed via successive RAFT polymerizations for the synthesis of star polymers with variable arm numbers and lengths. The removal of Boc groups from the polymers results in water soluble pH-responsive cationic hyperbranched architectures with tuneable pH responsiveness, differing from 6.8–7.5 due to the incorporation of various degrees of hydrophobic chain end functionalities with the variation of monomer feed compositions. Dynamic light scattering (DLS), atomic force microscopy (AFM) and scanning electron microscopy (SEM) reveal the interesting self-assembly of the Boc-deprotected star polymers in aqueous media with amino acid-based cores and water soluble thermoresponsive arms. Below the hydrophilic to hydrophobic transition pH and temperature, star polymers remain as unimers in aqueous solution. However, above the transition pH (and below the transition temperature), they form multi-micellar aggregates, which further fuse together to form larger aggregates above the transition temperature.

QM/MM study of the reaction mechanism of the carboxyl transferase domain of pyruvate carboxylase from Staphylococcus aureus.

Pyruvate carboxylase (PC) catalyzes the carboxylation of pyruvate to produce oxaloacetate. Its activity is directly related to insulin release and thus PC has recently attracted great interest as a potential target for diabetes treatment. In this article, the catalytic mechanism of the carboxyl transferase domain of PC from Staphylococcus aureus was investigated by using a combined quantum-mechanical/molecular-mechanical approach. Our calculation results indicate that the catalytic reaction starts from the decarboxylation of carboxybiotin to generate an enol-BTI intermediate, followed by two consecutive proton-transfer processes (from T908 to enol-BTI and from PYR to T908). During the catalytic reaction, the main-chain amide of T908 plays a key role in catching CO2 and preventing its diffusion from the active center. A triad of residues, R571, Q575, and K741, contributes both to substrate binding and enol-pyruvate stabilization. The oxyanion hole, consisting of the side-chain hydroxyl of S911 and the side-chain amino of Q870, plays an important role in stabilizing the hydroxyl anion of BTI. The coordination of the metal cation by pyruvate is a second sphere, rather than an inner sphere, interaction, and the metal cation stabilizes the species through the medium of residue K741. The decarboxylation of carboxybiotin corresponds to the highest free energy barrier of 21.7 kcal/mol. Our results may provide useful information for both the regulation of enzyme activity and the development of related biocatalytic applications.

Polarimetry-controlled fluorescent color in oriented LC biopolyesters

Oriented films of the thermotropic liquid-crystalline (LC) polymer, poly{3-benzylidene amino-4-hydroxybenzoic acid (3,4-BAHBA)-co-trans-4-hydroxycinnamic acid (4HCA)} (Poly(3,4-BAHBA-co-4HCA)), were prepared by shearing the LC melt. X-Ray and polarized microscopy analyses indicated that the main chains were oriented perpendicularly to the shear direction, and the benzylidene amino side chains were parallely oriented. Furthermore, the oriented film showed polarized fluorescence in which the analyzer rotation changed the emission color when the polarizer was set perpendicular to the sheared direction. Open image in new window

Lysine-based chiral vesicles

Uni-lamellar and multi-lamellar vesicles were prepared by the enantiomers of a biological molecule, l-lysine or d-lysine, with a double-tail weak monoacid, di-(2-ethylhexyl) phosphoric acid (abbreviated as DEHPA), in water. With the addition of DEHPA to lysine aqueous solutions, ion-pairs are formed through the acid-base reaction between the lysine cations and DEHP− anions. The self-assembled vesicles were proved to be driven by the hydrogen bonding between the side-chain amino groups in lysine molecules and the polar groups of DEHP− species. The combination of DEHPA and lysine through electrostatic interactions and hydrogen bonding reduces the cross-sectional area of the hydrophilic groups, improving the surface activity and inducing a microstructural transition from primitive aggregates to micelles, and to vesicles in solution. Due to the chirality of the lysine molecules, the aggregates exhibited diverse chiral properties along with the microstructural transitions.

Directed evolution of human heavy chain variable domain (VH) using in vivo protein fitness filter.

Human immunoglobulin heavy chain variable domains (VH) are promising scaffolds for antigen binding. However, VH is an unstable and aggregation-prone protein, hindering its use for therapeutic purposes. To evolve the VH domain, we performed in vivo protein solubility selection that linked antibiotic resistance to the protein folding quality control mechanism of the twin-arginine translocation pathway of E. coli. After screening a human germ-line VH library, 95% of the VH proteins obtained were identified as VH3 family members; one VH protein, MG2x1, stood out among separate clones expressing individual VH variants. With further screening of combinatorial framework mutation library of MG2x1, we found a consistent bias toward substitution with tryptophan at the position of 50 and 58 in VH. Comparison of the crystal structures of the VH variants revealed that those substitutions with bulky side chain amino acids filled the cavity in the VH interface between heavy and light chains of the Fab arrangement along with the increased number of hydrogen bonds, decreased solvation energy, and increased negative charge. Accordingly, the engineered VH acquires an increased level of thermodynamic stability, reversible folding, and soluble expression. The library built with the VH variant as a scaffold was qualified as most of VH clones selected randomly were expressed as soluble form in E. coli regardless length of the combinatorial CDR. Furthermore, a non-aggregation feature of the selected VH conferred a free of humoral response in mice, even when administered together with adjuvant. As a result, this selection provides an alternative directed evolution pathway for unstable proteins, which are distinct from conventional methods based on the phage display.