Altered Protein Conformation Research Articles

Vitamin B12 modulates Parkinson's disease LRRK2 kinase activity through allosteric regulation and confers neuroprotection.

Missense mutations in Leucine-Rich Repeat Kinase 2 (LRRK2) cause the majority of familial and some sporadic forms of Parkinson's disease (PD). The hyperactivity of LRRK2 kinase induced by the pathogenic mutations underlies neurotoxicity, promoting the development of LRRK2 kinase inhibitors as therapeutics. Many potent and specific small-molecule LRRK2 inhibitors have been reported with promise. However, nearly all inhibitors are ATP competitive-some with unwanted side effects and unclear clinical outcome-alternative types of LRRK2 inhibitors are lacking. Herein we identify 5'-deoxyadenosylcobalamin (AdoCbl), a physiological form of the essential micronutrient vitamin B12 as a mixed-type allosteric inhibitor of LRRK2 kinase activity. Multiple assays show that AdoCbl directly binds LRRK2, leading to the alterations of protein conformation and ATP binding in LRRK2. STD-NMR analysis of a LRRK2 homologous kinase reveals the contact sites in AdoCbl that interface with the kinase domain. Furthermore, we provide evidence that AdoCbl modulates LRRK2 activity through disrupting LRRK2 dimerization. Treatment with AdoCbl inhibits LRRK2 kinase activity in cultured cells and brain tissue, and prevents neurotoxicity in cultured primary rodent neurons as well as in transgenic C. elegans and D. melanogaster expressing LRRK2 disease variants. Finally, AdoCbl alleviates deficits in dopamine release sustainability caused by LRRK2 disease variants in mouse models. Our study uncovers vitamin B12 as a novel class of LRRK2 kinase modulator with a distinct mechanism, which can be harnessed to develop new LRRK2-based PD therapeutics in the future.

The Crg1 N-Terminus Is Essential for Methyltransferase Activity and Cantharidin Resistance in Saccharomyces cerevisiae.

Crg1 is an S-adenosylmethionine (SAM)-dependent methyltransferase required for cantharidin resistance in yeast. Crg1 has a well-characterized methyltransferase domain that inactivates cantharidin by methylation. However, the remaining part of the Crg1 protein is yet to be functionally characterized. In this study, we identified an essential role of the N-terminus of Crg1 in methyltransferase activity and cantharidin resistance. Yeast cells lacking 41 residues of the N-terminus of Crg1 ( crg1ΔN) showed hypersensitivity to cantharidin as same as the null mutant, crg1. The mass spectrometry-based biochemical enzyme assay revealed a loss of methyltransferase activity in Crg1ΔN, which justifies the loss of cantharidin resistance, as well. The subcellular distribution of Crg1ΔN-daGFP showed cytoplasmic aggregates, whereas wild-type Crg1-daGFP was distributed normally in the cytoplasm. Interestingly, the Crg1-methyltransferase domain point mutants (D44A, D67A, and E105A/D108A) also showed the same cytoplasmic aggregates as Crg1ΔN-daGFP. In silico prediction of the tertiary structures of these mutants indicated an altered protein conformation. Altogether, these observations suggest that the N-terminal truncation, as well as the point mutations in the methyltransferase domain, alters the native folding of Crg1 methyltransferase, resulting in a loss of enzyme activity. Furthermore, the crg1ΔN mutant showed the same phenotypes as the crg1 null mutant in the presence of cantharidin, i.e., lethal cell growth, PE auxotrophy, temperature sensitivity, endoplasmic reticulum stress, GPI anchor missorting, and cell wall damage. Overall, this study identifies an essential role of the N-terminus of Crg1 in methyltransferase activity and cantharidin resistance.

Plasmon‐Enhanced Fluorescent Sensor based on Aggregation‐Induced Emission for the Study of Protein Conformational Transformation

AbstractThe alteration in protein conformation not only affects the performance of its biological functions, but also leads to a variety of protein‐mediated diseases. Developing a sensitive strategy for protein detection and monitoring its conformation changes is of great significance for the diagnosis and treatment of protein conformation diseases. Herein, a plasmon‐enhanced fluorescence (PEF) sensor is developed, based on an aggregation‐induced emission (AIE) molecule to monitor conformational changes in protein, using prion protein as a model. Three anthracene derivatives with AIE characteristics are synthesized and a water‐miscible sulfonate salt of 9,10‐bis(2‐(6‐sulfonaphthalen‐2‐yl)vinyl)anthracene (BSNVA) is selected to construct the PEF–AIE sensor. The sensor is nearly non‐emissive when it is mixed with cellular prion protein while emits fluorescence when mixed with disease‐associated prion protein (PrPSc). The kinetic process of conformational conversion can be monitored through the fluorescence changes of the PEF–AIE sensor. By right of the amplified fluorescence signal, this PEF–AIE sensor can achieve a detection limit 10 pM lower than the traditional AIE probe and exhibit a good performance in human serum sample. Furthermore, molecular docking simulations suggest that BSNVA tends to dock in the β‐sheet structure of PrP by hydrophobic interaction between BSNVA and the exposed hydrophobic residues.

Protein conformational alterations induced by the retinal excited state in proton and sodium pumping rhodopsins.

Retinal proteins' biological activity is triggered by the retinal chromophore's light absorption, which initiates a photocycle. However, the mechanism by which retinal light excitation induces the protein's response is not completely understood. Recently, two new retinal proteins were discovered, namely, King Sejong 1-2 (KS1-2) and Nonlabens (Donghaeana) dokdonensis (DDR2), which exhibit H+ and Na+ pumping activities, respectively. To pinpoint whether protein conformation alterations can be achieved without light-induced retinal C13[double bond, length as m-dash]C14 double-bond isomerization, we utilized the hydroxylamine reaction, which cleaves the protonated Schiff base bond through which the retinal chromophore is covalently bound to the protein. The reaction is accelerated by light even though the cleavage is not a photochemical reaction. Therefore, the cleavage reaction may serve as a tool to detect protein conformation alterations. We discovered that in both KS1-2 and DDR2, the hydroxylamine reaction is light accelerated, even in artificial pigments derived from synthetic retinal in which the crucial C13[double bond, length as m-dash]C14 double-bond isomerization is prevented. Therefore, we propose that in both proteins the light-induced retinal charge redistribution taking place in the retinal excited state polarizes the protein, which, in turn, triggers protein conformation alterations. A further general possible application of the present finding is associated with other photoreceptor proteins having retinal or other non-retinal chromophores whose light excitation may affect the protein conformation.

Rational Side-Chain Amino Acid Substitution in Firefly Luciferase for Improved Thermostability

Improving the thermostability of the luciferase from firefly (Photinus pyralis) needs to be solved to broaden its industrial applications. In this paper, molecular dynamic (MD) simulations were used to identify 4 amino acid substitutions (P183V, E325K, Q338V, and E354K) which might have a significant influence on the thermostability of luciferase. Root-mean-square deviation values were calculated to further evaluate the effect of these mutations on thermostability of the enzyme and demonstrated that the thermostability of the corresponding protein variants was in the order E354K > E325K > WT > P183V > Q338V. Following the MD simulation, the enzyme variants were expressed in a recombinant host, and the results showed that the t1/2, T50, and Tm of mutant E354K were increased 2.32-fold, and 4.5 and 3.3°C more compared with the wild type, respectively. MD simulations, as well as circular dichroism and fluorescence spectroscopy were further applied to elucidate the conformational differences between the wild-type and E354K luciferases. The results indicated that a possible explanation for the improved thermostability of E354K enzyme lies in the formation of a salt bridge between Lys354 and Glu311 and alteration of protein conformation.

Calcium-Binding Proteins with Disordered Structure and Their Role in Secretion, Storage, and Cellular Signaling

Calcium is one of the most important second messengers and its intracellular signaling regulates many aspects of cell physiology. Calcium ions, like phosphate ions, are highly charged and thus are able to alter protein conformation upon binding; thereby they constitute key factors in signal transduction. One of the most common calcium-binding structural motifs is the EF-hand, a well-defined helix-loop-helix structural domain, present in many calcium-binding proteins (CBPs). Nonetheless, some CBPs contain non-canonical, disordered motifs, which usually bind calcium with high capacity and low affinity, and which represent a subset of proteins with specific functions, but these functions rarely involve signaling. When compared with phosphorylation-mediated signal transduction, the role of intrinsic disorder in calcium signaling is significantly less prominent and not direct. The list of known examples of intrinsically disordered CBPs is relatively short and the disorder in these examples seems to be linked to secretion and storage. Calcium-sensitive phosphatase calcineurin is an exception, but it represents an example of transient disorder, which is, nevertheless, vital to the functioning of this protein. The underlying reason for the different role of disordered proteins in the two main cellular signaling systems appears to be linked to the gradient of calcium concentration, present in all living cells.

A brief overview of the Swi1 prion-[SWI+

Prion and prion-like phenomena are involved in the pathology of numerous human neurodegenerative diseases. The budding yeast, Saccharomyces cerevisiae, has a number of endogenous yeast prions-epigenetic elements that are transmitted as altered protein conformations and often manifested as heritable phenotypic traits. One such yeast prion, [SWI+], was discovered and characterized by our laboratory. The protein determinant of [SWI+], Swi1 was found to contain an amino-terminal, asparagine-rich prion domain. Normally, Swi1 functions as part of the SWI/SNF chromatin remodeling complex, thus, acting as a global transcriptional regulator. Consequently, prionization of Swi1 leads to a variety of phenotypes including poor growth on non-glucose carbon sources and abolishment of multicellular features-with implications on characterization of [SWI+] as being detrimental or beneficial to yeast. The study of [SWI+] has revealed important knowledge regarding the chaperone systems supporting prion propagation as well as prion-prion interactions with [PSI+] and [RNQ+]. Additionally, an intricate regulatory network involving [SWI+] and other prion elements governing multicellular features in yeast has begun to be revealed. In this review, we discuss the current understanding of [SWI+] in addition to some possibilities for future study.

Structural underpinnings of oestrogen receptor mutations in endocrine therapy resistance.

Oestrogen receptor-α (ERα), a key driver of breast cancer, normally requires oestrogen for activation. Mutations that constitutively activate ERα without the need for hormone binding are frequently found in endocrine-therapy-resistant breast cancer metastases and are associated with poor patient outcomes. The location of these mutations in the ER ligand-binding domain and their impact on receptor conformation suggest that they subvert distinct mechanisms that normally maintain the low basal state of wild-type ERα in the absence of hormone. Such mutations provide opportunities to probe fundamental issues underlying ligand-mediated control of ERα activity. Instructive contrasts between these ERα mutations and those that arise in the androgen receptor (AR) during anti-androgen treatment of prostate cancer highlight differences in how activation functions in ERs and AR control receptor activity, how hormonal pressures (deprivation versus antagonism) drive the selection of phenotypically different mutants, how altered protein conformations can reduce antagonist potency and how altered ligand-receptor contacts can invert the response that a receptor has to an agonist ligand versus an antagonist ligand. A deeper understanding of how ligand regulation of receptor conformation is linked to receptor function offers a conceptual framework for developing new anti-oestrogens that might be more effective in preventing and treating breast cancer.

Codon Optimization in the Production of Recombinant Biotherapeutics: Potential Risks and Considerations.

Biotherapeutics are increasingly becoming the mainstay in the treatment of a variety of human conditions, particularly in oncology and hematology. The production of therapeutic antibodies, cytokines, and fusion proteins have markedly accelerated these fields over the past decade and are probably the major contributor to improved patient outcomes. Today, most protein therapeutics are expressed as recombinant proteins in mammalian cell lines. An expression technology commonly used to increase protein levels involves codon optimization. This approach is possible because degeneracy of the genetic code enables most amino acids to be encoded by more than one synonymous codon and because codon usage can have a pronounced influence on levels of protein expression. Indeed, codon optimization has been reported to increase protein expression by> 1000-fold. The primary tactic of codon optimization is to increase the rate of translation elongation by overcoming limitations associated with species-specific differences in codon usage and transfer RNA (tRNA) abundance. However, in mammalian cells, assumptions underlying codon optimization appear to be poorly supported or unfounded. Moreover, because not all synonymous codon mutations are neutral, codon optimization can lead to alterations in protein conformation and function. This review discusses codon optimization for therapeutic protein production in mammalian cells.

Pin1 Is Involved in HDAC6-mediated Cancer Cell Motility.

Histone deacetylase 6 (HDAC6), a member of the HDAC enzymes, has been reported to play substantial roles in many cellular processes. Evidence shows that deregulation of HDAC6 may be involved in the progression of some cancers, neurodegenerative diseases, and inflammatory disorders. However, little is known regarding the effect of post-translational modification of HDAC6 on cellular localization and biological functions. In the present study, we identified four phosphorylation sites on HDAC6 under normal conditions by mass spectrometry analysis. Two phosphorylation sites, pSer22 and pSer412, are recognized as Pin1 (peptidyl-prolyl cis/trans isomerase NIMA-interacting 1) substrates. Pin1 can interact with HDAC6 and be involved in HDAC6-mediated cell motility. Pin1 depletion abrogates HDAC6-induced cell migration and invasion in H1299 lung cancer cells. The findings of this study suggest that Pin1 might regulate HDAC6-mediated cell motility through alteration of protein conformation and function. Our results indicate the complexity of activity regulation between HDAC6 and Pin1, expanding knowledge regarding the multifunctional roles of Pin1 in tumorigenesis and cancer progression.

Computational Modeling of Lysine Post-Translational Modification: An Overview

Living organisms have a magnificent ordered and complex structure. In regulating the cellular functions, post-translational modifications (PTMs) are critical molecular measures. They alter protein conformation, modulating their activity, stability and localization. Up to date, more than 300 types of PTMs are experimentally discovered in vivo and in vitro pathways [1,2]. Major and common PTMs are methylation, ubiquitination, succinylation, phosphorylation, glycosylation, acetylation, and sumoylation. PTM is a biological mechanism common to both prokaryotic and eukaryotic organisms, which controls the protein functions and stability or the proteolytic cleavage of regulatory subunits and affects all aspects of cellular life. The PTM of a protein can also determine the cell signaling state, turnover, localization, and interactions with other proteins [3]. Therefore, the analysis of proteins and their PTMs are particularly important for the study of heart disease, cancer, neurodegenerative diseases and diabetes [4,5]. Since the characterization of PTMs gets invaluable insight into the cellular functions in etiological processes, there are still challenges. Specifically, the major challenges in studying PTMs are the development of specific detection and purification methods.

Retinal Binding to Apo-Gloeobacter Rhodopsin: The Role of pH and Retinal-Carotenoid Interaction.

Over the past few decades, the structure, functions, properties, and molecular mechanisms of retinal proteins have been studied extensively. The newly studied retinal protein Gloeobacter rhodopsin (gR) acts as a light-driven proton pump, transferring a proton from the cytoplasmic region to the extracellular region of a cell following light absorption. It was previously shown that gR can bind the carotenoid salinixanthin (sal). In the present study, we report the effect of pH on the binding of retinal to the apo-protein of gR, in the presence and absence of sal, to form the gR pigment. We found that binding at different pH levels reflects the titration of two different protein residues, one at the lower pKa 3.5 and another at the higher pKa 8.4, that affect the pigment's formation. The maximum amount of pigment was formed at pH 5, both with and without the presence of sal. The introduction of sal accelerates the rate of pigment formation by a factor of 190. Furthermore, it is suggested that occupation of the binding site by the retinal chromophore induces protein conformational alterations which in turn affect the carotenoid conformation, which precedes the formation of the retinal-protein covalent bond. Our examination of synthetic retinal analogues in which the ring structure was modified revealed that, in the absence of sal, the retinal ring structure affects the rate of pigment formation and that the intact structure is needed for efficient pigment formation. However, the presence of sal abolishes this effect, and all-trans retinal and its modified ring analogues bind at a similar rate.

Structural and Functional Properties Changes of β-Conglycinin Exposed to Hydroxyl Radical-Generating Systems.

The objective of the present study was to examine the structural and functional changes of β-conglycinin exposed to oxidizing radicals produced by FeCl3/H2O2/ascorbic acid hydroxyl radical-generating system (HRGS) for 3 h at room temperature. Increasing H2O2 concentrations resulted in a loss of histidine residues, lysine residues, and available lysine, which was accompanied by the formation of protein carbonyls and disulphide bonds (p < 0.05). Changes in secondary structure, surface hydrophobicity, and intrinsic fluorescence indicated that hydroxyl radicals had induced protein unfolding and conformational alterations. Results from SDS-PAGE implied that a small amount of protein cross-linkages produced by oxidative incubation. The emulsifying properties of β-conglycinin were gradually improved with the increasing extent of oxidation. The structural changes above contributed to the reduction of potential allergenicity of β-conglycinin, as verified by specific ELISA analysis. These results suggest that moderate oxidation could partially improve the protein functional properties and reduced the potential allergy of protein, providing guidance for effective use of moderately oxidized soy protein in the industry.

Cytoskeleton and nucleoskeleton involvement in processes of cytomixis in plants.

Cytomixis is a form of cell-to-cell nuclear migration that involves the interaction of dynamic cytoskeletal components with the nucleus through signalling systems and linker complexes. In cytomixis two known mechanisms can be involved: actomyosin and/or microtubules and their associated motors. Perinuclear actin anchors and determines the direction of nuclear movement. In microsporogenesis cytomixis is probably initiated by a cascade of signals that trigger prophase reorganization of nucleus and cytoskeleton, and is a result of cytoskeletal protein activation, as well as a weakening of mechanisms responsible for anchoring the nucleus. The interactions between nuclei and the cytoskeleton are mediated by linker complexes that play a major role in nuclear positioning and shape, chromatin-nuclear envelope interactions, nucleoskeleton organization, gene expression and genome organization. Other contributing factors include changes in the protein composition and post-translational modifications that alter protein conformation. Cytomixis appears also to have relevance to higher order structuring, influencing tissue and organ architecture, causing collective forms of cell interactions and information exchange within a single continuum. In this review we summarize our current understanding of the cytoskeleton dynamic function in cytomictic nuclear migration.

Solvent sensitivity of protein aggregation in Cu, Zn superoxide dismutase: a molecular dynamics simulation study

Misfolding and aggregation of Cu, Zn Superoxide Dismutase (SOD1) is often found in amyotrophic lateral sclerosis (ALS) patients. The central apo SOD1 barrel was involved in protein maturation and pathological aggregation in ALS. In this work, we employed atomistic molecular dynamics (MD) simulations to study the conformational dynamics of SOD1barrel monomer in different concentrations of trifluoroethanol (TFE). We find concentration dependence unusual structural and dynamical features, characterized by the local unfolding of SOD1barrel. This partially unfolded structure is characterized by the exposure of hydrophobic core, is highly dynamic in nature, and is the precursor of aggregation seen in SOD1barrel. Our computational studies supports the hypothesis of the formation of aggregation ‘building blocks’ by means of local unfolding of apo monomer as the mechanism of SOD1 fibrillar aggregation. The non-monotonic TFE concentration dependence of protein conformational changes was explored through simulation studies. Our results suggest that altered protein conformation and dynamics within its structure may underlie the aggregation of SOD1 in ALS.

Mutations in membrane cofactor protein (CD46) gene in Indian children with hemolytic uremic syndrome.

BackgroundMutations in the CD46 gene account for an important proportion of patients with atypical hemolytic uremic syndrome (aHUS) who characteristically show multiple relapses, no response to plasma exchange and low recurrence risk in allograft. We screened for mutations in CD46 in patients with and without circulating anti-factor H (FH) antibodies–associated aHUS.MethodsWe estimated CD46 surface expression by flow cytometry and sequenced the CD46 gene in 23 and 56 patients with and without circulating anti-FH antibodies, respectively. Human Splicing Finder and PolyPhen2 were used for in silico prediction of pathogenicity.ResultsTwo novel and three known (c.286 +2T > G, c.104G > A and c.565T > G) mutations in CD46 were found in nine (11.4%) patients; one patient had a variant of unknown significance and two patients presented during the first year of life. Novel intronic (c.1127 + 46C > G) and exonic (c.911C > T) mutations are proposed to activate cryptic splicing sites or alter protein conformation. Markedly reduced CD46 surface expression was found in homozygous states in five patients.ConclusionPatients with mutations in CD46 present at all ages, including the first year of life. Mutations in intron 2, (c.286 +2T > G) may be a potential hot spot in Indian children. Flow cytometry for CD46 expression is a satisfactory screening tool enabling early diagnosis.

The herpevac trial for women: Sequence analysis of glycoproteins from viruses obtained from infected subjects.

The Herpevac Trial for Women revealed that three dose HSV-2 gD vaccine was 58% protective against culture-positive HSV-1 genital disease, but it was not protective against HSV-2 infection or disease. To determine whether vaccine-induced immune responses had selected for a particular gD sequence in strains infecting vaccine recipients compared with viruses infecting control subjects, genetic sequencing studies were carried out on viruses isolated from subjects infected with HSV-1 or HSV-2. We identified naturally occurring variants among the gD sequences obtained from 83 infected subjects. Unique or low frequency amino acid substitutions in the ectodomain of gD were found in 6 of 39 HSV-1-infected subjects and in 7 of 44 HSV-2-infected subjects. However, no consistent amino acid change was identified in isolates from gD-2 vaccine recipients compared with infected placebo recipients. gC and gE surround and partially shield gD from neutralizing antibody, and gB also participates closely in the viral entry process. Therefore, these genes were sequenced from a number of isolates to assess whether sequence variation may alter protein conformation and influence the virus strain’s capacity to be neutralized by vaccine-induced antibody. gC and gE genes sequenced from HSV-1-infected subjects showed more variability than their HSV-2 counterparts. The gB sequences of HSV-1 oral isolates resembled each other more than they did gB sequences rom genital isolates. Overall, however, comparison of glycoprotein sequences of viral isolates obtained from infected subjects did not reveal any singular selective pressure on the viral cell attachment protein or surrounding glycoproteins due to administration of gD-2 vaccine.

PTI-125 binds and reverses an altered conformation of filamin A to reduce Alzheimer's disease pathogenesis

We show that amyloid-β1-42 (Aβ42) triggers a conformational change in the scaffolding protein filamin A (FLNA) to induce FLNA associations with α7-nicotinic acetylcholine receptor (α7nAChR) and toll-like receptor 4 (TLR4). These aberrant associations respectively enable Aβ42's toxic signaling via α7nAChR to hyperphosphorylate tau protein, and TLR4 activation to release inflammatory cytokines. PTI-125 is a small molecule that preferentially binds altered FLNA and restores its native conformation, restoring receptor and synaptic activities and reducing its α7nAChR/TLR4 associations and downstream pathologies. Two-month oral PTI-125 administration to triple-transgenic (3xTg) Alzheimer's disease (AD) mice before or after apparent neuropathology and to 8-month wildtypes with milder neuropathologies reduced receptor dysfunctions and improved synaptic plasticity, with some improvements in nesting behavior and spatial and working memory in 3xTg AD mice. PTI-125 also reduced tau hyperphosphorylation, aggregated Aβ42 deposition, neurofibrillary tangles, and neuroinflammation. Efficacy in postmortem AD and Aβ42-treated age-matched control hippocampal slices was concentration-dependent starting at 1 picomolar (pM) concentration. PTI-125 is the first therapeutic candidate to preferentially bind an altered protein conformation and reverse this proteopathy.

Protein conjugation with PAMAM nanoparticles: Microscopic and thermodynamic analysis

PAMAM dendrimers form strong protein conjugates that are used in drug delivery systems. We report the thermodynamic and binding analysis of polyamidoamine (PAMAM-G4) conjugation with human serum albumin (HSA), bovine serum albumin (BSA) and milk beta-lactoglobulin (b-LG) in aqueous solution at physiological pH. Hydrophobicity played a major role in PAMAM-protein interactions with more hydrophobic b-LG forming stronger polymer-protein conjugates. Thermodynamic parameters showed PAMAM-protein bindings occur via hydrophobic and H-bonding contacts for b-LG, while van der waals and H-bonding interactions prevail in HSA and BSA-polymer conjugates. The protein loading efficacy was 45–55%. PAMAM complexation induced major alterations of protein conformation. TEM images show major polymer morphological changes upon protein conjugation.

Folic acid delivery by serum proteins: loading efficacy and protein morphology

The loading efficacy of folic acid with serum proteins human serum albumin (HSA), bovine serum albumin (BSA), and beta-lactoglobulin (β-LG) was analyzed and the effect of acid conjugation on protein morphology was determined. Structural analysis showed that folic acid binds HSA, BSA, and β-LG via hydrophilic, hydrophobic, and H-bonding contacts with BSA forming more stable conjugates than HSA and β-LG. Molecular modeling showed the presence of several H-bonding systems, stabilizing acid–protein conjugates. Folic acid conjugation alters protein conformation by major alterations of α-helix and β-sheet. TEM images showed major protein morphological changes inducing protein aggregation upon acid interaction. The results show that serum proteins can deliver folic acid to target molecules.