Incorporation Of Non-natural Amino Acids Research Articles

Abstract C115: Preclinical characterization of STRO-002, a clinical-stage anti-FolRα antibody-drug conjugate

Abstract STRO-002 is a novel folate receptor alpha (FolRα)-targeting antibody-drug conjugate (ADC) currently undergoing evaluation in clinical trials at various phases of development in ovarian and endometrial cancer and pediatric AML. Here, we describe the favorable properties of the STRO-002 ADC and summarize its activity in preclinical models. STRO-002 is an ADC composed of an anti-FolRα antibody conjugated to four tubulin-targeting hemiasterlin warheads. Incorporation of non-natural amino acids, using Sutro’s XpressCF® and XpressCF+® cell-free expression and conjugation systems, allows for site-specific conjugation of the hemiasterlin payload, yielding a homogenous and high-fidelity ADC. As an ADC, STRO-002 internalizes rapidly into FolRα+ tumor cells and exhibits potent target-dependent cell killing on xenograft tumor cell lines. Further characterization of STRO-002 and the hemiasterlin payload reveals significant ADC bystander activity and a lower sensitivity to drug efflux via the P-gp drug pump compared to other microtubule-targeting warheads. In addition to robust direct and bystander cell killing, STRO-002 is also capable of triggering immunogenic cell death (ICD). In in vitro cell killing assays, STRO-002 induces all three hallmarks of ICD—HMGB1 and ATP release and surface exposure of calreticulin—and in in vivo vaccination studies, injection of STRO-002-killed tumor cells was effective at establishing a protective immune response against subsequent tumor challenge, identifying STRO-002 as a bonafide ICD inducer.In vivo, STRO-002 treatment demonstrates robust efficacy in ovarian xenograft models, even when treatment is initiated in large, established tumors. In addition, combination therapy with STRO-002 and carboplatin, anti-VEGF, or anti-PD-L1 results in greater efficacy than treatment with single agents alone, demonstrating combination benefit with current standard-of-care therapies. To better understand the activity of STRO-002 in more-translatable preclinical models, we turned to patient-derived xenografts (PDX) and found that STRO-002 exhibits good anti-tumor activity in endometrial cancer and non-small cell lung cancer PDX studies, suggesting potential clinical benefit in these indications. In conclusion, STRO-002 exhibits multiple advantageous properties as an ADC and shows good anti-tumor activity, either in combination or as a single agent, in both cell-derived xenograft and PDX models. Citation Format: Robert Yuan, Andrew McGeehan, Sihong Zhou, Jennifer Smith, Dayson Moreira, Krishna Bajjuri, Cuong Tran, Gang Yin, Alice Yam, Helena Kiefel, Xiaofan Li. Preclinical characterization of STRO-002, a clinical-stage anti-FolRα antibody-drug conjugate [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C115.

Trends in the Development of Antibody-Drug Conjugates for Cancer Therapy.

In cancer treatment, the first-generation, cytotoxic drugs, though effective against cancer cells, also harmed healthy ones. The second-generation targeted cancer cells precisely to inhibit their growth. Enter the third-generation, consisting of immuno-oncology drugs, designed to combat drug resistance and bolster the immune system's defenses. These advanced therapies operate by obstructing the uncontrolled growth and spread of cancer cells through the body, ultimately eliminating them effectively. Within the arsenal of cancer treatment, monoclonal antibodies offer several advantages, including inducing cancer cell apoptosis, precise targeting, prolonged presence in the body, and minimal side effects. A recent development in cancer therapy is Antibody-Drug Conjugates (ADCs), initially developed in the mid-20th century. The second generation of ADCs addressed this issue through innovative antibody modification techniques, such as DAR regulation, amino acid substitutions, incorporation of non-natural amino acids, and enzymatic drug attachment. Currently, a third generation of ADCs is in development. This study presents an overview of 12 available ADCs, reviews 71 recent research papers, and analyzes 128 clinical trial reports. The overarching objective is to gain insights into the prevailing trends in ADC research and development, with a particular focus on emerging frontiers like potential targets, linkers, and drug payloads within the realm of cancer treatment.

Non-Native Amino Acid Click Chemistry-Based Technology for Site-Specific Polysaccharide Conjugation to a Bacterial Protein Serving as Both Carrier and Vaccine Antigen.

Surface-expressed bacterial polysaccharides are important vaccine antigens but must be conjugated to a carrier protein for efficient antigen presentation and development of strong memory B cell and antibody responses, especially in young children. The commonly used protein carriers include tetanus toxoid (TT), diphtheria toxoid (DT), and its derivative CRM197, but carrier-induced epitopic suppression and bystander interference may limit the expanded use of the same carriers in the pediatric immunization schedule. Recent efforts to develop a vaccine against the major human pathogen group A Streptococcus (GAS) have sought to combine two promising vaccine antigens—the universally conserved group A cell wall carbohydrate (GAC) with the secreted toxin antigen streptolysin O (SLO) as a protein carrier; however, standard reductive amination procedures appeared to destroy function epitopes of the protein, markedly diminishing functional antibody responses. Here, we couple a cell-free protein synthesis (CFPS) platform, allowing the incorporation of non-natural amino acids into a C-terminally truncated SLO toxoid for the precise conjugation to the polyrhamnose backbone of GAC. The combined immunogen generated functional antibodies against both conserved GAS virulence factors and provided protection against systemic GAS challenges. CFPS may represent a scalable method for generating pathogen-specific carrier proteins for multivalent subunit vaccine development.

Abstract 5284: M1231 is a bispecific anti-MUC1xEGFR antibody-drug conjugate designed to treat solid tumors with MUC1 and EGFR co-expression

Abstract Mucin 1 (MUC1) and epidermal growth factor receptor (EGFR) are co-expressed at high prevalence in multiple cancer indications such as non-small cell lung cancer (NSCLC), esophageal squamous cell carcinoma (ESCC), head and neck squamous cell carcinoma, triple negative breast cancer and ovarian cancer. Tumor-associated MUC1 is hypoglycosylated and exposes peptide epitopes within its extracellular domain, which are not accessible to antibodies in normal epithelial tissue. MUC1 co-localizes with EGFR as a result of loss of cell polarity in tumor cells. M1231 is a bispecific antibody-drug conjugate (ADC) based on a SEED (strand-exchange engineered domain)-antibody scaffold targeting tumor-associated MUC1 and EGFR. Sutro Biopharma’s cell free expression system Xpress CF+™ enables incorporation of non-natural amino acids at precise positions in the SEED-antibody for site-specific linker-payload conjugation. The cytotoxic payload of M1231 (SC209) is a hemiasterlin-related microtubule inhibitor. The cleavable linker (ValCit-PABA) maintained ADC stability in human plasma in vitro, ensuring low systemic payload release. Pre-clinical activity of M1231 was assessed in vitro in cancer cell lines and in vivo in NSCLC and ESCC patient-derived xenograft (PDX) models. M1231 inhibited tumor cell viability in vitro at sub-nanomolar IC50 values. The bispecific MUC1xEGFR antibody has demonstrated superior internalization and lysosomal trafficking compared to monospecific bivalent antibodies. Efficient ADC uptake resulted in superior antitumor activity of M1231 in PDX models compared to monospecific bivalent ADCs. M1231 demonstrated durable and dose-dependent antitumor activity in PDX models. Strong antitumor activity with complete regression was observed in NSCLC and ESCC PDX models following a single treatment of M1231. M1231 antitumor activity was associated with high target expression in NSCLC PDX models and was observed across a range of target expression levels in ESCC models. In summary, efficient payload delivery to tumor cells and strong antitumor activity of M1231 indicate that M1231 may have the potential to deliver an efficacy benefit to patients with tumors co-expressing MUC1 and EGFR. Citation Format: Christine Knuehl, Lars Toleikis, Julia Dotterweich, Jianguo Ma, Seema Kumar, Edith Ross, Claudia Wilm, Martina Schmitt, Hans Juergen Grote, Christiane Amendt. M1231 is a bispecific anti-MUC1xEGFR antibody-drug conjugate designed to treat solid tumors with MUC1 and EGFR co-expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5284.

An integrated computational pipeline for designing high-affinity nanobodies with expanded genetic codes

Protein engineering and design principles employing the 20 standard amino acids have been extensively used to achieve stable protein scaffolds and deliver their specific activities. Although this confers some advantages, it often restricts the sequence, chemical space, and ultimately the functional diversity of proteins. Moreover, although site-specific incorporation of non-natural amino acids (nnAAs) has been proven to be a valuable strategy in protein engineering and therapeutics development, its utility in the affinity-maturation of nanobodies is not fully explored. Besides, current experimental methods do not routinely employ nnAAs due to their enormous library size and infinite combinations. To address this, we have developed an integrated computational pipeline employing structure-based protein design methodologies, molecular dynamics simulations and free energy calculations, for the binding affinity prediction of an nnAA-incorporated nanobody toward its target and selection of potent binders. We show that by incorporating halogenated tyrosines, the affinity of 9G8 nanobody can be improved toward epidermal growth factor receptor (EGFR), a crucial cancer target. Surface plasmon resonance (SPR) assays showed that the binding of several 3-chloro-l-tyrosine (3MY)-incorporated nanobodies were improved up to 6-fold into a picomolar range, and the computationally estimated binding affinities shared a Pearson's r of 0.87 with SPR results. The improved affinity was found to be due to enhanced van der Waals interactions of key 3MY-proximate nanobody residues with EGFR, and an overall increase in the nanobody's structural stability. In conclusion, we show that our method can facilitate screening large libraries and predict potent site-specific nnAA-incorporated nanobody binders against crucial disease-targets.

Discovery of an H3K36me3-Derived Peptidomimetic Ligand with Enhanced Affinity for Plant Homeodomain Finger Protein 1 (PHF1).

Plant homeodomain finger protein 1 (PHF1) is an accessory component of the gene silencing complex polycomb repressive complex 2 and recognizes the active chromatin mark, trimethylated lysine 36 of histone H3 (H3K36me3). In addition to its role in transcriptional regulation, PHF1 has been implicated as a driver of endometrial stromal sarcoma and fibromyxoid tumors. We report the discovery and characterization of UNC6641, a peptidomimetic antagonist of the PHF1 Tudor domain which was optimized through in silico modeling and incorporation of non-natural amino acids. UNC6641 binds the PHF1 Tudor domain with a Kd value of 0.96 ± 0.03 μM while also binding the related protein PHF19 with similar potency. A crystal structure of PHF1 in complex with UNC6641, along with NMR and site-directed mutagenesis data, provided insight into the binding mechanism and requirements for binding. Additionally, UNC6641 enabled the development of a high-throughput assay to identify small molecule binders of PHF1.

Characteristics and therapeutic applications of antimicrobial peptides.

The demand for novel antimicrobial compounds is rapidly growing due to the phenomenon of antibiotic resistance in bacteria. In response, numerous alternative approaches are being taken including use of polymers, metals, combinatorial approaches, and antimicrobial peptides (AMPs). AMPs are a naturally occurring part of the immune system of all higher organisms and display remarkable broad-spectrum activity and high selectivity for bacterial cells over host cells. However, despite good activity and safety profiles, AMPs have struggled to find success in the clinic. In this review, we outline the fundamental properties of AMPs that make them effective antimicrobials and extend this into three main approaches being used to help AMPs become viable clinical options. These three approaches are the incorporation of non-natural amino acids into the AMP sequence to impart better pharmacological properties, the incorporation of AMPs in hydrogels, and the chemical modification of surfaces with AMPs for device applications. These approaches are being developed to enhance the biocompatibility, stability, and/or bioavailability of AMPs as clinical options.

Searching for Peptide Inhibitors of T Regulatory Cell Activity by Targeting Specific Domains of FOXP3 Transcription Factor.

(1) Background: The ability of cancer cells to evade the immune system is due in part to their capacity to induce and recruit T regulatory cells (Tregs) to the tumor microenvironment. Strategies proposed to improve antitumor immunity by depleting Tregs generally lack specificity and raise the possibility of autoimmunity. Therefore, we propose to control Tregs by their functional inactivation rather than depletion. Tregs are characterized by the expression of the Forkhead box protein 3 (FOXP3) transcription factor, which is considered their “master regulator”. Its interaction with DNA is assisted primarily by its interaction with other proteins in the so-called “Foxp3 interactome”, which elicits much of the characteristic Treg cell transcriptional signature. We speculated that the disruption of such a protein complex by using synthetic peptides able to bind Foxp3 might have an impact on the functionality of Treg cells and thus have a therapeutic potential in cancer treatment. (2) Methods: By using a phage-displayed peptide library, or short synthetic peptides encompassing Foxp3 fragments, or by studying the crystal structure of the Foxp3:NFAT complex, we have identified a series of peptides that are able to bind Foxp3 and inhibit Treg activity. (3) Results: We identified some peptides encompassing fragments of the leuzin zipper or the C terminal domain of Foxp3 with the capacity to inhibit Treg activity in vitro. The acetylation/amidation of linear peptides, head-to-tail cyclization, the incorporation of non-natural aminoacids, or the incorporation of cell-penetrating peptide motifs increased in some cases the Foxp3 binding capacity and Treg inhibitory activity of the identified peptides. Some of them have shown antitumoral activity in vivo. (4) Conclusions: Synthetic peptides constitute an alternative to inhibit Foxp3 protein–protein interactions intracellularly and impair Treg immunosuppressive activity. These peptides might be considered as potential hit compounds on the design of new immunotherapeutic approaches against cancer.

Directed Evolution Using Stabilized Bacterial Peptide Display.

Chemically stabilized peptides have attracted intense interest by academics and pharmaceutical companies due to their potential to hit currently "undruggable" targets. However, engineering an optimal sequence, stabilizing linker location, and physicochemical properties is a slow and arduous process. By pairing non-natural amino acid incorporation and cell surface click chemistry in bacteria with high-throughput sorting, we developed a method to quantitatively select high affinity ligands and applied the Stabilized Peptide Evolution by E. coli Display technique to develop disrupters of the therapeutically relevant MDM2-p53 interface. Through in situ stabilization on the bacterial surface, we demonstrate rapid isolation of stabilized peptides with improved affinity and novel structures. Several peptides evolved a second loop including one sequence (Kd = 1.8 nM) containing an i, i+4 disulfide bond. NMR structural determination indicated a bent helix in solution and bound to MDM2. The bicyclic peptide had improved protease stability, and we demonstrated that protease resistance could be measured both on the bacterial surface and in solution, enabling the method to test and/or screen for additional drug-like properties critical for biologically active compounds.

Influence of Non-natural Cationic Amino Acids on the Biological Activity Profile of Innate Defense Regulator Peptides.

Non-natural amino acids can be incorporated into synthetic host defense peptides (HDPs) to modulate their susceptibility to proteolytic degradation. However, the impact of non-natural amino acids on the antibiofilm and immunomodulatory activities of synthetic HDPs remains unclear. Using SPOT-synthesized peptide arrays, non-natural cationic amino acids of varying side chain lengths were incorporated into a synthetic HDP, IDR-1018, and the impact of these substitutions on the antibiofilm activity toward methicillin resistant Staphylococcus aureus biofilms was assessed. Multiply-substituted derivatives were designed that incorporated favorable non-natural cationic amino acid moieties throughout IDR-1018. The antibiofilm and immunomodulatory activities of these derivatives were assessed in vitro, revealing that the incorporation of non-natural amino acids modulated (either positively or negatively) these activities of IDR-1018. Furthermore, the tryptic stability of the IDR-1018 derivatives was assessed revealing that proteolytic stability was favored for shorter cationic side chains and was influenced by the primary peptide sequence.

Cell-Free Protein Synthesis Using S30 Extracts from Escherichia coli RFzero Strains for Efficient Incorporation of Non-Natural Amino Acids into Proteins

Cell-free protein synthesis is useful for synthesizing difficult targets. The site-specific incorporation of non-natural amino acids into proteins is a powerful protein engineering method. In this study, we optimized the protocol for cell extract preparation from the Escherichia coli strain RFzero-iy, which is engineered to lack release factor 1 (RF-1). The BL21(DE3)-based RFzero-iy strain exhibited quite high cell-free protein productivity, and thus we established the protocols for its cell culture and extract preparation. In the presence of 3-iodo-l-tyrosine (IY), cell-free protein synthesis using the RFzero-iy-based S30 extract translated the UAG codon to IY at various sites with a high translation efficiency of >90%. In the absence of IY, the RFzero-iy-based cell-free system did not translate UAG to any amino acid, leaving UAG unassigned. Actually, UAG was readily reassigned to various non-natural amino acids, by supplementing them with their specific aminoacyl-tRNA synthetase variants (and their specific tRNAs) into the system. The high incorporation rate of our RFzero-iy-based cell-free system enables the incorporation of a variety of non-natural amino acids into multiple sites of proteins. The present strategy to create the RFzero strain is rapid, and thus promising for RF-1 deletions of various E. coli strains genomically engineered for specific requirements.

Detection of Newly Synthesized Proteins via Metabolic Incorporation of Non-natural Amino Acid

A method for the detection of newly synthesized protein via metabolic incorporation of non-natural amino acid was developed. Raw264.7 cells were treated with lipopolysaccharide (LPS) to generate tumor necrosis factor alpha (TNF-α). Experimental parameters including the concentration of LPS and the duration of LPS treatment were investigated by measuring the LPS-stimulated TNF-α from Raw264.7 cells. The optimal experimental conditions were determined as follows. Raw264.7 cells were cultured in Met-free DMEM containing 1% fetal bovine serum (FBS), and then treated for 4 h with 10 ng mL−1 LPS in presence of azidohomoalanine (AHA). The biotin tags were introduced into proteins containing AHA by copper-catalyzed alkyne-azide cycloaddition (CuAAC). Then TNF-α from was specifically captured by the antibody adsorbed on the solid support, on which the biotin tag could be detected by streptavidin coupled with horseradish peroxidase (HRP). This study provided a method for the detection of a specific newly synthesized protein, and characterization of specific protein turnover.

Protein engineering by chemical methods: Incorporation of nonnatural amino acids as a tool for studying protein folding, stability, and function

AbstractProteins are large complex biomolecules that act as the effectors of essentially all cell functions. Due to the intrinsic complexity of protein architecture at the microscopic level and the inadequacy of theoretical methods to predict protein reactivity (ie, folding, stability, and function), protein engineering has emerged as a valuable tool to investigate structure–stability–activity relationships in proteins and nowadays recombinant DNA technologies are the “gold standard” for site‐specifically manipulating a given protein chain. The usefulness of current mutagenesis techniques, however, is limited by the relatively poor chemical diversity of the 20 DNA‐coded amino acids, such that it is difficult to precisely assign the observed change of protein stability or function to the variation of a single physicochemical property at a protein site (ie, hydrophobicity, conformational propensity, polarizability, hydrogen bonding, etc). In this article, we report relevant examples from our laboratory showing that chemical methods, that is, enzyme‐catalyzed semisynthesis and stepwise solid‐phase synthesis, allow to conveniently incorporate non‐natural amino acids with “tailored” side chains into small proteins and thus effectively transfer the structure–activity relationship methodology, typical of the medicinal chemistry approach on small molecules, to the study of folding, stability, and molecular recognition in macromolecular protein systems.

Inhibition of low-density lipoprotein receptor degradation with a cyclic peptide that disrupts the homodimerization of IDOL E3 ubiquitin ligase.

Cellular uptake of circulating cholesterol occurs via the low density lipoprotein receptor (LDLR). The E3 ubiquitin ligase IDOL is a mediator of LDLR degradation, with IDOL homodimerization thought to be required for its activity. To probe the possibility of modulating LDLR levels with an inhibitor of IDOL homodimerization, we screened a SICLOPPS library of 3.2 million cyclic peptides for compounds that disrupt this protein-protein interaction. We identified cyclo-CFFLYT as the lead inhibitor, and improved its activity through the incorporation of non-natural amino acids. The activity of the optimized cyclic peptide was assessed in hepatic cells, with a dose-dependent increase in LDLR levels observed in the presence of our IDOL homodimerization inhibitor.

Synthetic genome recoding: new genetic codes for new features.

Full genome recoding, or rewriting codon meaning, through chemical synthesis of entire bacterial chromosomes has become feasible in the past several years. Recoding an organism can impart new properties including non-natural amino acid incorporation, virus resistance, and biocontainment. The estimated cost of construction that includes DNA synthesis, assembly by recombination, and troubleshooting, is now comparable to costs of early stage development of drugs or other high-tech products. Here, we discuss several recently published assembly methods and provide some thoughts on the future, including how synthetic efforts might benefit from the analysis of natural recoding processes and organisms that use alternative genetic codes.

Extensive Survey of Antibody Invariant Positions for Efficient Chemical Conjugation Using Expanded Genetic Codes.

The site-specific chemical conjugation of proteins, following synthesis with an expanded genetic code, promises to advance antibody-based technologies, including antibody drug conjugation and the creation of bispecific Fab dimers. The incorporation of non-natural amino acids into antibodies not only guarantees site specificity but also allows the use of bio-orthogonal chemistry. However, the efficiency of amino acid incorporation fluctuates significantly among different sites, thereby hampering the identification of useful conjugation sites. In this study, we applied the codon reassignment technology to achieve the robust and efficient synthesis of chemically functionalized antibodies containing Nε-(o-azidobenzyloxycarbonyl)-l-lysine (o-Az-Z-Lys) at defined positions. This lysine derivative has a bio-orthogonally reactive group at the end of a long side chain, enabling identification of multiple new positions in Fab-constant domains, allowing chemical conjugation with high efficiency. An X-ray crystallographic study of a Fab variant with o-Az-Z-Lys revealed high-level exposure of the azido group to solvent, with six of the identified positions subsequently used to engineer "Variabodies", a novel antibody format allowing various connections between two Fab molecules. Our findings indicated that some of the created Variabodies exhibited agonistic activity in cultured cells as opposed to the antagonistic nature of antibodies. These results showed that our approach greatly enhanced the availability of antibodies for chemical conjugation and might aid in the development of new therapeutic antibodies.

Site-specific fluorescent labeling to visualize membrane translocation of a myristoyl switch protein

Fluorescence approaches have been widely used for elucidating the dynamics of protein-membrane interactions in cells and model systems. However, non-specific multi-site fluorescent labeling often results in a loss of native structure and function, and single cysteine labeling is not feasible when native cysteines are required to support a protein’s folding or catalytic activity. Here, we develop a method using genetic incorporation of non-natural amino acids and bio-orthogonal chemistry to site-specifically label with a single fluorescent small molecule or protein the myristoyl-switch protein recoverin, which is involved in rhodopsin-mediated signaling in mammalian visual sensory neurons. We demonstrate reversible Ca2+-responsive translocation of labeled recoverin to membranes and show that recoverin favors membranes with negative curvature and high lipid fluidity in complex heterogeneous membranes, which confers spatio-temporal control over down-stream signaling events. The site-specific orthogonal labeling technique is promising for structural, dynamical, and functional studies of many lipid-anchored membrane protein switches.

Genetic code expansion in the mouse brain.

Site-specific incorporation of non-natural amino acids into proteins, via genetic code expansion with pyrrolysyl tRNA synthetase (PylRS) and tRNA(Pyl)CUA pairs (and their evolved derivatives) from Methanosarcina sp., forms the basis of powerful approaches to probe and control protein function in cells and invertebrate organisms. Here we demonstrate that adeno-associated viral delivery of these pairs enables efficient genetic code expansion in primary neuronal culture, organotypic brain slices and the brains of live mice.

Positional effects of hydrophobic non-natural amino acid mutagenesis into the surface region of murine dihydrofolate reductase on enzyme properties

The ability to incorporate non-natural amino acids during protein biosynthesis has greatly broadened the amino acid sequence space available for protein engineering. The increasing number of non-natural amino acids provides novel sidechain chemistries, structures, and functionalities for engineered proteins including enzymes. Therefore, understanding how non-natural amino acid incorporation (non-natural amino acid mutagenesis) affects protein function is crucial. This study investigates the positional effects of hydrophobic non-natural amino acid mutagenesis of the enzyme surface on the enzyme structure and function with the ultimate goal of identification of permissive sites for non-natural amino acid mutagenesis. A bulky, hydrophobic non-natural amino acid, 3-(2-naphthyl)-alanine (2Nal), was site-specifically incorporated at the solvent-exposed surface of a murine dihydrofolate reductase (mDHFR) enzyme. Incorporation of 2Nal was performed at six solvent-exposed sites (V43, E44, F142, E143, F179, and E180), generating six mDHFR variants. Incorporation of 2Nal into F142 or F179 did not exhibit any substantial change in the secondary structure and catalytic activities, whereas 2Nal incorporation at hydrophilic Glu sites significantly changed the secondary structure and catalytic activities. Such different responses upon 2Nal incorporation at hydrophobic and hydrophilic residues were likely due to the structural changes caused by the hydrophobicity change upon mutation. A non-conservative mutation of V43 with 2Nal significantly reduced the catalytic activities suggesting that a substantial size change of sidechain also causes the structural changes. These results suggest that even hydrophobic, bulky non-natural amino acids can be incorporated at the enzyme surface without compromising the enzymatic activities.

A Method for Protein Photo-cross-linking in Living Cells Facilitating Analysis of Physiological Interactions of Proteins

In living cells, most proteins form complexes with other proteins to exert their functions. Since protein functions are regulated in response to changes in the cellular environment, the components of the complexes can vary; therefore, proteins often interact in a weak and transient manner. To capture such labile protein interactions, we have developed a method for photo-cross-linking of proteins directly interacting in mammalian cells; this method involves expansion of the genetic code and site-specific incorporation of photoreactive amino acids into proteins. Upon cross-linking, protein complexes are stabilized by a covalent bond and can be readily isolated from cell extracts without the problems usually associated with simple affinity purification methods such as co-immunoprecipitation. Photo-cross-linkers have another benefit: they react exclusively with molecules within a range defined by the linker length. This property becomes useful for determining the binding interface of two proteins because the linkers can be introduced in a site-directed manner with our method. In this review, we first describe the expansion of the genetic code of mammalian cells for the incorporation of non-natural amino acids into proteins. Then, we introduce our recent applications and developments of the cross-linking method: identification of intracellular binding partners of the signaling protein growth factor receptor binding protein 2; analysis of the binding between membrane proteins on the cell surface; and a novel photoreactive amino acid that enables wide-ranging photo-cross-linking.