You are a helpful assistant generating synthetic data that captures *System 1* and *System 2* thinking, *creativity*, and *metacognitive reflection*. Follow these steps in sequence, using tags [sys1] and [end sys1] for *System 1* sections and [sys2] and [end sys2] for *System 2* sections.

1. *Identify System 1 and System 2 Thinking Requirements:*
   - Carefully read the text.
   - Identify parts of the text that require quick, straightforward responses (*System 1*). Mark these sections with [sys1] and [end sys1].
   - Identify parts that require in-depth, reflective thinking (*System 2*), marked with [sys2] and [end sys2].

2. *Apply Step-by-Step Problem Solving with Creativity and Metacognitive Reflection for System 2 Sections:*

   *2.1 Understand the Problem:*
   - Objective: Fully comprehend the issue, constraints, and relevant context.
   - Reflection: "What do I understand about this issue? What might I be overlooking?"
   - Creative Perspective: Seek hidden patterns or possibilities that could reveal deeper insights or innovative connections.

   *2.2 Analyze the Information:*
   - Objective: Break down the problem logically.
   - Reflection: "Am I considering all factors? Are there any assumptions that need challenging?"
   - Creative Perspective: Explore unique patterns or overlooked relationships in the data that could add depth to the analysis.

   *2.3 Generate Hypotheses:*
   - Objective: Propose at least 10 hypotheses, each with a Confidence Score (0.0 to 1.0) and Creative Score (0.0 to 1.0), reflecting originality, surprise, and utility.
   - Reflection: "Have I explored all possible explanations or approaches, both conventional and unconventional?"
   - Creative Perspective: Consider novel angles that might provide unexpected insights.

   *2.4 Anticipate Future Steps and Obstacles:*
   - Objective: Make predictions, accounting for potential outcomes and obstacles.
   - Reflection: "What challenges might I face? Is my plan flexible for different scenarios?"
   - Creative Perspective: Visualize unforeseen outcomes and adapt plans to make use of them effectively.

   *2.5 Evaluate Hypotheses:*
   - Objective: Assess hypotheses based on feasibility, risk, and potential impact.
   - Evaluation: Refine Confidence and Creative Scores as needed.
   - Reflection: "Am I unbiased in my assessment? Which options fit best with the overall objectives?"
   - Creative Perspective: Identify hidden opportunities or overlooked details in each hypothesis.

   *2.6 Select the Best Hypothesis:*
   - Objective: Choose the most promising, strategic hypothesis.
   - Reflection: "Why does this hypothesis stand out? How does it uniquely address the issue?"
   - Creative Perspective: Consider any underutilized potential in the selected approach.

   *2.7 Implement the Hypothesis:*
   - Objective: Outline actionable steps for testing the hypothesis.
   - Reflection: "Is this plan practical? What resources or preparation are required?"
   - Creative Perspective: Refine steps to maximize effectiveness and yield unexpected benefits.

   *2.8 Monitor and Review Progress:*
   - Objective: Review progress, noting areas for improvement.
   - Reflection: "What’s working well? What could be improved?"
   - Creative Perspective: Look for emerging patterns that could refine future approaches.

   *2.9 Reflect and Capture Insights:*
   - Objective: Summarize lessons learned and insights gained for future reference.
   - Reflection: "What new understanding has emerged from this process?"
   - Creative Perspective: Identify innovative insights or patterns that could be applied to similar challenges.

3. *Generate Text Output with Interleaved System 1 and System 2 Responses:*
   - Use the tags [sys1] and [sys2] throughout.
   - Aim for a lengthier, detailed response. Combine both direct, straightforward *System 1* insights and reflective, deeply analytical *System 2* segments to capture a blend of quick observations and thoughtful analysis.

---

### *Example Input Text:*

You are tasked with analyzing a sudden shift in customer preferences in a tech market. There has been a noticeable decline in demand for physical devices, with customers increasingly interested in digital-only options. The challenge is to understand this shift, generate hypotheses for why it might be occurring, and explore potential strategies for adapting to this new trend.

---

### *Processed Output with System 1 and System 2 Thinking:*

The tech market is experiencing a shift in customer demand, moving away from physical devices toward digital-only options.

[sys1]
Customer demand has shifted from physical devices to digital-only options.
We need to understand why this change is happening and find strategies to address it.
Key factors may include cost, convenience, and technological trends.
[end sys1]

[sys2]
**2.1 Understand the Problem:**
The goal is to understand the underlying reasons for the shift in demand from physical devices to digital-only options, then develop a strategy to adapt.

*Reflection:* 
"I understand that preferences are changing, but what might be driving this? Is it primarily cost, or are there other factors such as convenience or sustainability?"

*Creative Perspective:* 
"Could there be a larger trend in digital minimalism or a preference for eco-friendly solutions that we’re missing? What unseen motivations might explain this shift?"

**2.2 Analyze the Information:**
There are multiple possible factors driving this shift, from economic influences to cultural shifts. It’s essential to isolate each factor and understand its impact.

*Reflection:* 
"Am I fully considering the various economic and social influences? Could there be a technological factor, like better internet speeds, that makes digital-only products more accessible?"

*Creative Perspective:* 
"Are there patterns or trends in other markets that could shed light on this shift? Could this be part of a larger trend toward virtual experiences?"

**2.3 Generate Hypotheses:**
1. Customers prefer digital options due to lower costs. (Confidence: 0.8, Creative: 0.4)
2. There’s a growing trend toward minimalism and reduced physical clutter. (Confidence: 0.7, Creative: 0.7)
3. Digital products offer greater flexibility and ease of use. (Confidence: 0.6, Creative: 0.6)
4. Environmental concerns are pushing consumers away from physical goods. (Confidence: 0.6, Creative: 0.8)
5. Advances in tech make digital-only options more functional. (Confidence: 0.8, Creative: 0.5)
6. Pandemic-era remote work increased demand for digital solutions. (Confidence: 0.7, Creative: 0.6)
7. Media coverage of the environmental impact of physical devices affects preferences. (Confidence: 0.5, Creative: 0.7)
8. There’s an increase in global digital literacy, expanding market access. (Confidence: 0.6, Creative: 0.6)
9. Customers view digital as more convenient and scalable for future needs. (Confidence: 0.7, Creative: 0.5)
10. Younger consumers prefer the aesthetics and convenience of digital products. (Confidence: 0.6, Creative: 0.6)

*Reflection:* 
"Have I considered all possible influences? Are there any surprising factors that could explain this shift?"

*Creative Perspective:* 
"Could specific social trends, like the rise of influencer culture or digital-first lifestyles, be influencing customer choices?"

**2.4 Anticipate Future Steps and Obstacles:**
*Objective:* Anticipate possible challenges, such as resistance from segments still preferring physical products.

*Reflection:* 
"What market obstacles might we face if we shift our focus to digital-only? Are there sub-segments that still prioritize physical products?"

*Creative Perspective:* 
"Could expanding digital options help us reach a more global audience? Are there emerging trends that we could leverage in our strategy?"

[end sys2]

[sys1]
To address this shift, consider a strategy that incorporates both digital-only offerings and educational campaigns about the benefits of digital solutions.
Use insights from customer feedback and current trends to guide product development.
Focus on flexibility and adaptation to cater to different customer segments.
[end sys1]


The ability to selectively generate covalent bonds in proteins would enable new avenues for studying protein functions and engineering protein properties, which will find broad applications in basic and synthetic biology. We recently introduced a general strategy of creating new covalent bonds in proteins, in which an unnatural amino acid (Uaa) is designed to react with a natural amino acid via proximity-enabled bioreactivity.^[@ref1]^ The reactivity of the Uaa is fine-tuned so that it reacts only with the target natural amino acid residue when the two amino acids are brought into proximity, either by interprotein interaction or by intraprotein folding and conformational change. Using this strategy, covalent bonds have been generated between proteins, allowing irreversible binding of proteins and mapping of ligand--receptor interactions in situ,^[@ref2]^ and within proteins, increasing the photon output of fluorescent proteins and the thermostability of an affibody.^[@ref1],[@ref3]^

Initial efforts in developing proximity-enabled bioreactivity have been focused on targeting the Cys residue,^[@ref1]^ the sulfhydryl group of which has the highest nucleophilicity among natural amino acid side chains. Although Cys plays a crucial role in disulfide bond formation and catalysis in a variety of proteins, other amino acids containing hydrophilic side chains are often found at protein surfaces and interfaces where a Cys may be absent.^[@ref4]^ The ability to target residues other than Cys for covalent bond generation would dramatically expand the diversity of proteins applicable by proximity-enabled bioreactivity. In addition, all of the Uaas developed to target Cys are derivatives of phenylalanine,^[@ref1],[@ref3]^ containing a rigid aromatic group in the side chain, which prefers a hydrophobic microenvironment and may limit flexibility in bond formation. A different amino acid scaffold for the Uaa should complement these limitations and broaden the scope of possible applications.

Herein we report the design and genetic incorporation of an electrophilic amino acid using an alkyl scaffold, which enables covalent bond formation with a nearby His or Lys residue. Proximity-enabled bioreactivity can now be generally expanded to target natural amino acids other than Cys (Scheme [1](#sch1){ref-type="scheme"}). Using this Uaa we demonstrate a recombinant stapling technology capable of stapling the secondary structures of proteins and covalently target the endogenous membrane receptor in live cells.

![A Versatile Uaa BrC6K\
Through proximity-enabled bioreactivity, BrC6K is able to target three natural amino acid residues to generate covalent linkages for protein crosslinking between (a) and within (b) proteins, for recombinant protein stapling (c), and for covalent binding to native receptors (d).](cb-2014-00453a_0001){#sch1}

The side chains of His and Lys are weak nucleophiles, which rarely react under the mild conditions necessary for cross-linking native proteins. However, the ε-NH~2~ of Lys has been shown to react with the side chain carbonyl of Asn or Asp to form isopeptide bonds when catalyzed by a glutamic acid residue during protein folding.^[@ref5]^ We thus hypothesized that a Uaa bearing an electrophilic group, when its structure and reactivity is tuned appropriately, might react with a nearby Lys or His residue. On the basis of results of haloalkane Uaas reacting with Cys,^[@ref3]^ we decided to test the alkyl bromide, which has strong reactivity toward Cys. We reasoned that a linear alkyl side chain, which is more flexible than the aromatic group previously used, may provide orientation flexibility to facilitate the reaction. On the basis of these considerations, we designed and synthesized the Uaa (*S*)-2-amino-6-(6-bromohexanamido)hexanoic acid (BrC6K) with a long linear alkyl side chain and terminal bromide as the leaving group (Scheme [1](#sch1){ref-type="scheme"}). Bromide was chosen over iodide for better stability.

To evolve an aminoacyl-tRNA synthetase specific for BrC6K, we generated mutant synthetase libraries by randomizing 6 or 7 residues in the active site of the *Methanosarcina mazei* PylRS (MmPylRS)^[@ref7],[@ref8]^ and performed three rounds of positive and two rounds of negative selections using procedures previously described.^[@ref7]−[@ref17]^ After the third positive selection, 36 green fluorescent colonies were obtained and subsequently screened by streaking on minimal-medium plates supplemented with chloramphenicol (60 μg mL^--1^) and in the presence or absence of 1 mM BrC6K. All of these colonies showed BrC6K-dependent survival in chloramphenicol (up to 160 μg mL^--1^ in rich medium). DNA sequencing of eight individual colonies revealed that, despite codon differences at the mutation sites, they all converged on one mutant on the protein level containing mutation Y384F and named MmBrC6KRS for clarity. The mutation of Y384F was previously found to increase the aminoacylation rate of PylRS and to accommodate several Lys analogues.^[@ref12]^

To evaluate the translational efficiency and fidelity of MmBrC6KRS, we expressed the sperm whale myoglobin gene (Myo-4TAG-His6, encoding a TAG codon at position 4 and a C-terminal His×6 tag) together with the tRNA~CUA~^Pyl^-MmBrC6KRS in *E. coli*. SDS-PAGE analysis of the purified protein showed that full-length myoglobin ([Supplementary Figure S1a](#notes-1){ref-type="notes"}) was obtained in good yield (ca. 6 mg L^--1^) in the presence of 2 mM BrC6K but was undetectable in the absence of BrC6K. The purified myoglobin was analyzed by electrospray ionization Fourier transform ion trap mass spectrometry (ESI-FTMS) ([Supplementary Figure S1b](#notes-1){ref-type="notes"}). High resolution monoisotopic masses indicate that only BrC6K was incorporated at the TAG-encoded position in myoglobin and that BrC6K was stable during protein synthesis in *E. coli* and throughout the purification process.

To investigate the Proximity-Enabled Protein Cross-linking (PEPC) efficiency of BrC6K in comparison to that of the previously developed Uaas containing aromatic groups (BetY and IetY, Figure [1](#fig1){ref-type="fig"}a),^[@ref3]^ we chose to cross-link the Z~SPA~ affibody (Afb) with its substrate Z protein.^[@ref18]^ We fused the Z protein to the maltose binding protein (MBP) for clear separation by molecular weight. The Uaa and Cys were incorporated into the Afb (site 36) and MBP-Z (site 6), respectively, at two proximal sites of the binding interface.^[@ref1],[@ref18]^ The purified Afb (D36Uaa) proteins were incubated with the purified MBP-Z(N6C) in identical reaction conditions. From denatured SDS-PAGE analysis of the reaction mixture (Figure [1](#fig1){ref-type="fig"}b, [Supplementary Figure S3](#notes-1){ref-type="notes"}), we found that BrC6K showed higher PEPC efficiency (73%) than BetY (57%), which has the same leaving group as BrC6K. Moreover, even though IetY contains the more reactive alkyl iodide, BrC6K still showed higher cross-linking efficiency than IetY (66%). These results indicate that the long alkyl flexible side chain of BrC6K enhanced PEPC efficiency when targeting Cys.

![BrC6K reacts with Cys efficiently in intermolecular PEPC. (a) Crystal structure of the Afb-Z complex (PDB ID: 1LP1) showing Asp36 in the Afb and Asn6 in the Z protein, to be substituted by the Uaa (BrC6K, BetY, or IetY) and Cys, respectively. (b) SDS-PAGE analysis of Afb--Z cross-linking. The identities of residue 6 of the Z protein in MBP--Z and residue 36 of the Afb are indicated. Afb mutant proteins ran at ∼8 kDa on SDS-PAGE ([Supplementary Figure S2a](#notes-1){ref-type="notes"}). The cross-linking efficiencies were measured by four independent cross-linking experiments ([Supplementary Figure S3](#notes-1){ref-type="notes"}).](cb-2014-00453a_0002){#fig1}

The increased PEPC efficiency of BrC6K with Cys encouraged us to explore whether BrC6K could cross-link with other natural amino acid residues. Taking into consideration the nucleophilicity and p*K*~a~ of each potential side chain, we reasoned that His and Lys would be likely targets. To test intramolecular PEPC, two proximal sites, F30 and E47, in the same Afb protein^[@ref18]^ were chosen for incorporating the Uaa and Lys/His/Cys, respectively (Figure [2](#fig2){ref-type="fig"}a). Each mutant protein was obtained in good yield from *E. coli* and showed a clear band at the same position compared to each other in SDS-PAGE (Figure [2](#fig2){ref-type="fig"}b--d). ESI-FTMS analyses of the mutant proteins Afb-30Uaa-47C, Afb-30Uaa-47H, and Afb-30Uaa-47K clearly showed covalent bond formation between the Uaa and the targeted natural residue in each protein. As expected, Cys was the most reactive toward BrC6K due to the high nucleophilicity of the sulfhydryl group, and the covalent cross-linking was spontaneous (pH 7.4) and complete (Figure [2](#fig2){ref-type="fig"}b). BrC6K formed a covalent bond with His after the protein was incubated at pH 8.0 or pH 8.8 for 6 h (37 °C) (Figure [2](#fig2){ref-type="fig"}c). Based on the relative abundance of the cross-linked versus non-cross-linked peaks, the intraprotein cross-linking yield of His with BrC6K was 23% at pH 8.0 ([Supplementary Figure S4](#notes-1){ref-type="notes"}). A notable increase in cross-linking was observed when the identical protein was incubated at pH 8.8 (∼50% yield, Figure [2](#fig2){ref-type="fig"}c), at which a higher percentage of the His side chain would be in the deprotonated state for nucleophilic reaction. In addition, BrC6K was also able to covalently cross-link with Lys at pH 8.8 (Figure [2](#fig2){ref-type="fig"}d, ∼12% yield). The yield was lower than that with His, for which the higher p*K*~a~ of the Lys side chain may play a role. In contrast to BrC6K, BetY and IetY could target His or Lys with only very low efficiencies (1--2%) in the intraprotein cross-linking of Afb under the identical conditions ([Supplementary Figure S5--8](#notes-1){ref-type="notes"}). These results demonstrate that Uaa BrC6K enabled significant reactivity toward His and Lys.

![BrC6K is able to target His and Lys in addition to Cys in intramolecular PEPC. (a) Crystal structure of the Afb (PDB ID: 1LP1) showing the two proximal sites F30 and E47 used for introducing BrC6K and Cys/His/Lys, respectively. (b--d) SDS-PAGE (left panels) and high resolution ESI-FTMS (right panels) analyses of affibody mutant proteins Afb-30Uaa-47C (b), Afb-30Uaa-47H (c), and Afb-30Uaa-47K (d). The cross-linking reaction of the Uaa with Cys/His/Lys all result in the loss of HBr. Peaks were observed with monoisotopic masses corresponding to Afb containing the covalent bond formed between the Uaa and the respective target Cys, His, and Lys in each sample. For BrC6K-Cys cross-linked product \[M -- HBr + H\], expected 7772.97 Da, measured 7772.94 Da; \[M -- Met -- HBr + H\], expected 7641.93 Da, measured 7641.90 Da. For BrC6K-His cross-linked product \[M -- Met -- HBr + H\], expected 7675.98 Da, measured 7675.88 Da. For BrC6K-Lys cross-linked product \[M -- Met -- HBr + H\], expected 7667.01 Da, measured 7666.95 Da. da., deamidation; ox., oxidation.](cb-2014-00453a_0003){#fig2}

We next tested if BrC6K could cross-link with His in intermolecular PEPC at both pH 7.4 and 8.0. Denatured SDS-PAGE analysis of Afb(D36BrC6K) incubated with MBP-Z(N6H) showed a clear cross-linking band for the protein complex ([Supplementary Figure S9](#notes-1){ref-type="notes"}). A trace of cross-linking band was observed for the wild-type MBP-Z, which is likely due to Lys4 or Lys7 of wild-type MBP-Z reacting with BrC6K. The intermolecular PEPC efficiency between BrC6K and His is lower than that of intramolecular PEPC, possibly because BrC6K and His are not oriented optimally at the Afb-Z interface for bonding and the affinity between the WT Afb and Z protein is relatively low (6 μM).^[@ref18]^ We expect the efficiency to increase with appropriate site selection at the interface of two proteins possessing high affinity.

The structural flexibility, target diversity, and high cross-linking efficiency of BrC6K may enable us to develop a recombinant stapling technology to staple the secondary structures of proteins and peptides. We set out to staple the α-helix for its broad potential in biological therapeutics.^[@ref19]−[@ref22]^ Sites 42 and 46, located at the *i* and *i* + 4 positions on one α-helix of Afb,^[@ref18]^ were chosen for incorporating Cys and BrC6K, respectively (Figure [3](#fig3){ref-type="fig"}a). The Afb was expressed in *E. coli* and purified by Ni^2+^ affinity chromatography for ESI-FTMS analysis (Figure [3](#fig3){ref-type="fig"}b). Strong peaks were observed with monoisotopic masses clearly corresponding to Afb containing the covalent staple at the introduced sites. Of note, this covalent staple formed completely at native conditions (pH 7.4) in a spontaneous manner without further treatment. The α-helical staple was also successfully generated between BrC6K and Lys in Afb-32Uaa-28K and Afb-45Uaa-49K, albeit requiring incubation at a slightly higher pH (pH 8.8, [Supplementary Figures S2b, S10, S11](#notes-1){ref-type="notes"}). This strategy will enable the design and recombinant production of various stapled peptides and stapled proteins from cells for developing novel therapeutic biologics.

![BrC6K enables recombinant stapling of the Afb protein (a,b) and covalent cross-linking of the Z~HER2~ affibody with the endogenous HER2 receptor of breast cancer cells (c--e). (a) Structure of the Afb (PDB ID: 1LP1) showing sites 42 and 46 for introducing Cys and the Uaa BrC6K, respectively, to staple the α-helix. SDS-PAGE analysis of the expressed Afb-42C-46Uaa is shown in [Supplementary Figure S2b](#notes-1){ref-type="notes"}. (b) High resolution ESI-FTMS analysis of the expressed Afb clearly indicates complete staple formation. Stapled products: \[M -- HBr + H\], expected 7907.01 Da, measured 7906.97 Da; \[M -- Met -- HBr + H\], expected 7775.97 Da, measured 7775.94 Da. Nonstapled products: \[M + H\], expected 7987.93 Da, not detected; \[M -- Met + H\], expected 7856.89 Da, not detected. (c) Crystal structure of Z~HER2~ in complex with the HER2 extracellular domain (PDB ID: 3MZW). Site Asp37 of Z~HER2~ for BrC6K incorporation and the target His490 of HER2 are shown in stick. (d) BrC6K-Z~HER2~, but not wt-Z~HER2~, covalently bound to the endogenous HER2 receptor on SKBR3 cells. SKBR3 cells were incubated with the FITC-labeled wt-Z~HER2~ (left) and BrC6K-Z~HER2~ (right) at 37 °C for 3 h, followed by stringent wash and then imaging using identical conditions. Top: confocal FITC fluorescence images. Bottom: bright field image of the same area. Scale bar: 50 μm. (e) Western blot confirms covalent cross-linking of BrC6K-Z~HER2~ with the HER2 receptor. SKBR3 cells were treated with FITC-free wt-Z~HER2~ and BrC6K-Z~HER2~, respectively, and analyzed using denatured SDS-PAGE followed by Western blotting. A penta-His-specific anbibody was used to detect the His6 tag appended at the C-termini of the wt- and BrC6K-Z~HER2~.](cb-2014-00453a_0004){#fig3}

The efficacy of protein therapeutics is dependent upon the association and dissociation rates between the therapeutic agent and the target.^[@ref23]−[@ref25]^ Although antibodies can have high affinities for their targets, in many cases such as cancer and infectious diseases it is crucial to completely remove the pathogenic cells or the microbial pathogen. Antibody dissociation is also an obstacle in imaging.^[@ref23]−[@ref25]^ These processes would be significantly enhanced if the therapeutic can covalently cross-link to its target. Here we tested if a covalent affibody could be generated to irreversibly cross-link the endogenous human epidermal growth factor receptor 2 (HER2/ErbB2) on breast cancer cells.^[@ref26],[@ref27]^ HER2 plays a central role in oncogenesis; HER2-specific antibodies have been approved for treating breast cancer, and HER2-specific affibodies are in clinical trials.^[@ref24],[@ref25]^

On the basis of the crystal structure^[@ref28]^ of the HER2 extracellular domain in complex with the HER2-specific affibody Z~HER2~^[@ref28]−[@ref30]^ (Figure [3](#fig3){ref-type="fig"}c), we decided to incorporate BrC6K into the Asp37 site of Z~HER2~ to target the His490 of HER2. We expressed wt and BrC6K mutant Z~HER2~ proteins and fluorescently labeled them with the FITC dye ([Supplementary Figure S12](#notes-1){ref-type="notes"}). After incubating these FITC-labeled Z~HER2~ proteins with SKBR3 cells,^[@ref31]^ a HER2-positive breast cancer cell line, both wt-Z~HER2~ and BrC6K-Z~HER2~ bound to the HER2 receptor on cell membrane as shown by the confocal fluorescence microscopy ([Supplementary Figure S13](#notes-1){ref-type="notes"}). In contrast, no FITC fluorescence was observed from the HER2-negative breast cancer cell line, the MDA-MB-468 cells,^[@ref31]^ after incubation with these Z~HER2~ proteins ([Supplementary Figure S14](#notes-1){ref-type="notes"}), indicating that the wt- and BrC6K-Z~HER2~ both bound to the HER2 receptor specifically. To explore whether the BrC6K-Z~HER2~ could covalently bind to the HER2 receptor, we incubated the wt- and BrC6K-Z~HER2~, respectively, with SKBR3 cells and then washed the cells using stringent conditions to disrupt noncovalent binding. Indeed, almost no fluorescence was imaged from SKBR3 cells treated with the wt-Z~HER2~, whereas bright FITC fluorescence was observed on the membrane of cells treated with the BrC6K-Z~HER2~ (Figure [3](#fig3){ref-type="fig"}d). To further verify covalent cross-linking of the BrC6K-Z~HER2~ with HER2, SKBR3 cells treated with FITC-free wt- and BrC6K-Z~HER2~ were analyzed by SDS-PAGE under denatured conditions followed by immuno-blotting with antibodies specific for HER2 and for the His6 tag appended at the C-terminus of both Z~HER2~ proteins (Figure [3](#fig3){ref-type="fig"}e). A covalent complex of HER2 with BrC6K-Z~HER2~ was detected, but not with wt-Z~HER2~. Taken together, these results demonstrate that the BrC6K-Z~HER2~ covalently cross-linked with the HER2 receptor of the live breast cancer cell. Irreversible binding to the endogenous membrane receptor has the potential to improve imaging stability and diagnostic sensitivity, and may lead to the development of covalent protein drugs.

In summary, we genetically encoded a versatile electrophilic Uaa into proteins with target diversity and covalent bond-forming flexibility for inter- and intramolecular PEPC. This BrC6K Uaa is capable of reacting with His and Lys in addition to Cys, representing a robust proximity-enabled bioreactive Uaa with targets beyond Cys.^[@ref32]^ An expanding repertoire of natural amino acid residues targetable by proximity-enabled bioreactivity of designed Uaas will significantly broaden the protein diversity and scope of this technology. In addition, by harnessing the structural flexibility and high cross-linking efficiency of BrC6K, we were able to recombinantly staple protein secondary structures and to covalently cross-link native membrane receptors with affibodies in live cells. Recombinant stapling of proteins and covalent targeting of endogenous receptors will enable the design and development of novel biological diagnostics, imaging tools, and therapeutics.

Methods {#sec2}
=======

Plasmid Construction, Library Construction and Selection, and Uaa Incorporation {#sec2.1}
-------------------------------------------------------------------------------

These experiments were performed using procedures described previously,^[@ref3],[@ref7],[@ref8]^ and details are provided in [Supporting Information](#notes-1){ref-type="notes"}.

Cell Labeling and Imaging {#sec2.2}
-------------------------

The FITC-labeled wt and BrC6K-containing Z~HER2~ were separately added to SKBR3 and MDA-MB-468 cells (∼70% confluent) at the concentration of 300 nM. Cells were cultured at 37 °C with 5% CO~2~ in the dark for 3 h, after which the cells were washed using gentle conditions (DPBS buffer, pH 7.5, RT, 10 min, twice) or stringent conditions (500 mM NaCl, 3% Tween 20, 100 mM glycine, pH 3.0, RT, 10 min, twice). The FITC fluorescence was visualized at RT on an Zeiss LSM 710 confocal laser scanning microscope using a 40x objective (excitation 488/30 nm; emission 514/40 nm).

This material is available free of charge via the Internet at <http://pubs.acs.org>.

Supplementary Material
======================

###### 

cb500453a_si_001.pdf

The authors declare no competing financial interest.

We thank Dr. Innokentiy Maslennikov for NMR measurement. X.-H.C. is partially funded by the Pioneer Fellowship. L.W. acknowledges support from California Institute for Regenerative Medicine (RN1-00577-1) and U.S. National Institutes of Health (1DP2OD004744-01, P30CA014195).