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]


Published: March 6, 2012

Introduction {#sec1}
============

The conformational diseases are characterized by conformational transitions in mutant proteins that allow multimerization and aberrant protein deposition. They include Alzheimer\'s disease and Lewy body dementia, the prion encephalopathies, systemic amyloidosis, and the serpinopathies ([@bib24]). The serpinopathies result from the polymerization of mutants of members of the serine protease inhibitor or serpin superfamily of proteins ([@bib14]). The archetype of the serpinopathies is α~1~-antitrypsin deficiency. α~1~-antitrypsin is synthesized by hepatocytes ([@bib23]) and released into the circulation, where it protects the lung from the actions of neutrophil elastase ([@bib16]). Severe α~1~-antitrypsin deficiency is found in 1:2000 individuals of North European descent ([@bib2]) and typically results from homozygosity for the Z allele (Glu342Lys). The Z mutation causes α~1~-antitrypsin to adopt an intermediate conformation and rapidly polymerize ([Figure 1](#fig1){ref-type="fig"}A; [@bib25]). Polymers accumulate within the endoplasmic reticulum of hepatocytes in association with neonatal hepatitis, cirrhosis, and hepatocellular carcinoma. Concomitant deficiency of circulating α~1~-antitrypsin predisposes to severe, early onset emphysema ([@bib13]).

Previous data suggest that serpin polymers are formed by intermolecular linkage of the reactive center loop of one molecule with β sheet A of another ([@bib9; @bib15; @bib19; @bib25; @bib36]). However, a recent model suggests that the polymer is linked by a more extensive domain swap involving both the reactive center loop and strand 5 of β sheet A ([@bib22; @bib41]). The controversy may be partly explained by our recent finding that structurally distinct polymers are favored by different conditions used to induce polymerization in vitro ([@bib10]). Resolving these issues at a residue-specific level is important for targeting drug design against the pathological polymerization of α~1~-antitrypsin deficiency. We have therefore used nuclear magnetic resonance (NMR) spectroscopy and a deficiency-associated and polymerogenic α~1~-antitrypsin variant to characterize structural and dynamic changes associated with population of the pathological intermediate state.

Results {#sec2}
=======

Detection of α~1~-Antitrypsin~Queen\'s~ {#sec2.1}
---------------------------------------

A mutation that, to our knowledge, has not previously been described was detected in an individual whose circulating α~1~-antitrypsin levels (median value 0.6 mg/ml; normal range 1.5--3.5 mg/ml) were between those expected of a Z homozygote and an MZ heterozygote. Genotyping revealed a compound heterozygote for the Z allele and a Lys154Asn variant that we have named α~1~-antitrypsin~Queen\'s~.

Population of the Pathological Intermediate {#sec2.2}
-------------------------------------------

Recombinant Lys154Asn α~1~-antitrypsin was characterized biochemically and biophysically ([Figure S1](#app2){ref-type="sec"}, available online; [Figures 1](#fig1){ref-type="fig"}B and 1C). The variant had 64% of the functional activity of wild-type α~1~-antitrypsin (i.e., 1.6-fold greater stoichiometry of inhibition \[SI\]) with an apparent association rate constant (k~app\'~) with bovine α-chymotrypsin of 5.5 (+/−1.1) × 10^4^ M^-1^s^-1^ (n = 3). When corrected for the increase in SI, the association rate constant (k~app~) for Lys154Asn α~1~-antitrypsin was 1.3 × 10^5^ M^-1^s^-1^, an order of magnitude lower than the wild-type control (1.16 × 10^6^ M^-1^s^-1^). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) confirmed increased substrate-like behavior in Lys154Asn α~1~-antitrypsin relative to the wild-type ([Figure S1](#app2){ref-type="sec"}C).

The Queen\'s variant formed polymers under physiological conditions ([Figure 1](#fig1){ref-type="fig"}B). This was slow at 37°C, and more rapid at 42°C and higher temperatures. The general pathological relevance of these polymers was demonstrated by the presence of the 2C1 neo-epitope that is specific for polymers associated with disease ([@bib29]; [Figure 1](#fig1){ref-type="fig"}B, right).

Polymer formation occurs via population of an intermediate state ([@bib6]). We therefore studied intermediate formation by Lys154Asn α~1~-antitrypsin. The variant populated intermediate states more readily than wild-type α~1~-antitrypsin in urea-induced and thermal denaturing conditions ([Figure S1](#app2){ref-type="sec"}A; [Figure 1](#fig1){ref-type="fig"}C). Since we had established the disease relevance of the polymerogenic intermediate at temperatures in the physiological range, we focused on structural changes induced by incubation in these conditions. CD spectroscopy studies ([Figures 1](#fig1){ref-type="fig"}C and [S1](#app2){ref-type="sec"}D) indicated that the mutant fold underwent a subtle structural transition between 35°C and 45°C before more dramatic transition between 50°C and 60°C. Increasing concentration did not affect the first transition but caused the second transition to commence at a lower temperature. Wild-type α~1~-antitrypsin remained stable to 60°C and underwent a sharp unfolding transition at higher temperatures, consistent with previous studies ([@bib17; @bib31]). Increasing concentration cold-shifted the second half of the transition but did not affect its initial phase ([Figure S1](#app2){ref-type="sec"}D). Increasing protein concentration 2.5-fold (0.4 to 1.0 mg/ml) reduced the midpoint of the overall transition for wild-type α~1~-antitrypsin by ∼1.5°C and that of Lys154Asn α~1~-antitrypsin by ∼1.0°C. The profiles of the first part of the transition observed for wild-type α~1~-antitrypsin and the first transition observed for Lys154Asn α~1~-antitrypsin are both concentration-independent. Therefore, this likely reports upon structural change within the molecule. The subsequent transition is concentration-dependent. It therefore likely reports, to some degree, upon polymerization of a species populated during the first phase.

Polymerogenic intermediate formation in α~1~-antitrypsin has been previously studied by ANS fluorescence. ANS fluoresces strongly upon binding to regions in which both polar and hydrophobic motifs are exposed to solvent and so binds to intermediate states, rather than fully folded or unfolded polypeptides. The ability of the Queen\'s variant of α~1~-antitrypsin to bind ANS was therefore assessed ([Figure S1](#app2){ref-type="sec"}E). Lys154Asn α~1~-antitrypsin showed hyperfluorescence compared with the wild-type protein at 25°C. This was unexpected, since assays directly reporting structural information (CD spectroscopy, intrinsic fluorescence, and NMR spectroscopy) strongly indicated that both proteins were similarly well-folded and stable at this temperature. These observations of Lys154Asn α~1~-antitrypsin behavior are similar to those described for Z α~1~-antitrypsin ([@bib21]). Moreover, incubating Lys154Asn α~1~-antitrypsin with ANS for longer resulted in even greater hyperfluorescence, whereas increasing temperature to 37°C did not enhance this further ([Figure S1](#app2){ref-type="sec"}E). Wild-type α~1~-antitrypsin showed no change in ANS fluorescence at either temperature or upon prolonged incubation. These data indicate that for α~1~-antitrypsin variants in which conversion to the polymerogenic intermediate in solution is facile, ANS binding stabilizes the intermediate state sufficiently to skew the position of the native-intermediate equilibrium to the right. For Lys154Asn α~1~-antitrypsin, this effect is so significant that it outweighs any effect of temperature change between 25°C and 37°C. This supports our general view that the degree to which assays perturb solution equilibria must be considered when studying intermediate formation.

We hypothesized that ion-mobility mass spectrometry (IM-MS) might have sufficient sensitivity to detect population of the intermediate while minimally perturbing the solution equilibrium. At 20°C and 34°C, monomeric Lys154Asn α~1~-antitrypsin was indistinguishable from the wild-type protein by IM-MS. However, at 39°C, there was a 7.8% increase in the collision cross-section (CCS) of monomeric Lys154Asn α~1~-antitrypsin relative to the wild-type protein in keeping with population of an intermediate state ([Figure 1](#fig1){ref-type="fig"}C). The CCS of the intermediate state has been calculated as ∼18% greater than the native state, so a 7.8% increase in CCS is consistent with substantial population (∼40%) of the intermediate state in equilibrium at physiological temperatures.

NMR Spectroscopic Characterization of α~1~-Antitrypsin Solution Behavior and Mutation Effects {#sec2.3}
---------------------------------------------------------------------------------------------

We have observed and assigned backbone resonances for almost all residues in wild-type α~1~-antitrypsin in a series of NMR experiments (Biological Magnetic Resonance Bank entry 17804; [@bib30]), allowing us to analyze its solution behavior ([Figures 2](#fig2){ref-type="fig"} and [S2](#app2){ref-type="sec"}). Chemical shift index analysis ([@bib1]) was performed for Cα backbone bond signals alone or in conjunction with side-chain signals ([Figure S2](#app2){ref-type="sec"}A). This method did not use any crystallographically derived predictions, so it permitted residue-specific comparison of the secondary structure behavior of the protein directly observed in solution with that observed crystallographically. Although most of the secondary structure behavior of individual residues was conserved between solution and the crystal lattice conditions, β sheet C showed considerable lability in solution. The solution behavior of helices C and I was reported as α-helical by Cα backbone bond signals. However, when side-chain signals were also considered, these reported random coil behavior. Such differences may indicate that these motifs also displayed some lability in solution ([Figure S2](#app2){ref-type="sec"}A).

Two-dimensional (2D) NMR spectroscopy of ^15^N-labeled Lys154Asn α~1~-antitrypsin was then performed ([Figure 2](#fig2){ref-type="fig"}). At 25°C the well-dispersed regions of the spectrum contained a similar number (148) of cross-peaks to the number seen for the wild-type protein (152), and mean cross-peak intensities were similar for both proteins. A subset of 123 residues could be confidently assigned in the well-dispersed region of the ^1^H-^15^N TROSY-HSQC spectrum of Lys154Asn α~1~-antitrypsin. Overall peak integrals in the central region were also similar between the spectra for wild-type and Lys154Asn α~1~-antitrypsin ([Figure S2](#app2){ref-type="sec"}B). Nevertheless intensity and chemical shift differences were induced by the presence of the mutation in the native state of the protein ([Figure S2](#app2){ref-type="sec"}C). The degree of variation reported by intensity and chemical shift in the observed residues correlated closely. The magnitude of changes are shown by heatmap coloring of spheres mapped to the crystal structure and demonstrate the mutation most affects residues around the F-helix, strands 1--3 of β sheet A, and the reactive loop. In particular, the spectral effects were most dramatic on residues around hF and s3A, where the chemical shift changes were too great to allow their confident assignment in the mutant protein ([Figure S2](#app2){ref-type="sec"}C, black spheres).

NMR Spectral Changes and Polymerogenicity {#sec2.4}
-----------------------------------------

Our principal aim was to assess changes in α~1~-antitrypsin associated with population of the disease-relevant intermediate state. Native state behavior was defined as that observed for wild-type α~1~-antitrypsin at the temperatures studied in which no polymerization was observed. At 37°C, slow, irreversible, and linear loss of the observable, monomeric sample due to polymerization was observed in Lys154Asn but not the wild-type α~1~-antitrypsin. The linearity indicated a constant rate of polymer formation and hence that the population of the polymerogenic intermediate was also constant, that is, the solution was in pseudo-equilibrium. ^1^H-^15^N TROSY-HSQC spectra reported residue-specific structural and dynamic changes in Lys154Asn α~1~-antitrypsin by nonuniform intensity change in assigned cross-peaks relative to wild-type α~1~-antitrypsin ([Figure 3](#fig3){ref-type="fig"}A). Values are scaled for monomer concentration; they take into account changes observed between wild-type and mutant protein at 25°C in the absence of polymerization and changes observed in wild-type α~1~-antitrypsin between 25°C and 37°C. They therefore report differential changes in Lys154Asn α~1~-antitrypsin associated with population of the polymerogenic state.

Most reporter cross-peaks signaled minimal change in Lys154Asn α~1~-antitrypsin at 37°C ([Figure 3](#fig3){ref-type="fig"}B). Those in strand 5 in β sheet A, helix I, and the connecting linker strongly preserved intensity ([Figure 3](#fig3){ref-type="fig"}C). NMR intensity data are highly sensitive reporters of changes in structure and dynamics, so relatively large changes may be induced by quite small changes in solution behavior. Nevertheless, residues reporting the most dramatic intensity change in the polymerogenic ensemble notably occur in regions previously linked with conformational change (upper s3A, upper hF, underlying the upper s4A site, and hA; [@bib4; @bib26; @bib43]).

A further 12 distinct cross-peaks (signal:noise 11.5) were observed in the well-dispersed region of the spectrum in Lys154Asn but not the wild-type α~1~-antitrypsin at 37°C ([Figure S3](#app2){ref-type="sec"}A). The new cross-peaks disappeared on cooling and so may represent residues within a nonnative monomeric state, rather than polymers, since these did not dissociate (and would predictably give peaks of very low intensity). If so, these data support a process of native-non-native monomeric conformational exchange that is slow (≥ms) by NMR timescales, consistent with previous data ([@bib6]).

The mutation will mildly perturb local electrostatic interactions involving Lys154 ([Figure S3](#app2){ref-type="sec"}B). Its effects on resonances of nearby residues were too great to dissect by cross-peak analysis. We therefore studied a eukaryotic cell model of disease ([@bib17]) by mutagenesis. These clarified that the hydrogen bond of Lys154 (F-helix) with the main-chain carbonyl oxygen of Lys174 (neighboring linker) regulated against polymer formation ([Figure S3](#app2){ref-type="sec"}B).

The residue-specific changes caused by the Lys154Asn mutation differed markedly from those observed in urea conditions causing a similar degree of polymerization (1.5 M; [Figure S3](#app2){ref-type="sec"}C). Both patterns differ from that indicated by studies in guanidine or low pH conditions ([@bib22; @bib41]). Thus, the polymerogenic monomer ensemble to which this mild deficiency mutation reduces the energetic barrier, differs structurally and/or dynamically from polymerogenic ensembles induced by other conditions. The resultant polymers are also structurally distinct ([@bib10]).

Discussion {#sec3}
==========

Studying key native to intermediate transitions can elucidate mechanisms of the conformational diseases and aid targeting of novel therapeutic strategies. However, the intrinsic tendency of intermediates to adopt more stable, polydisperse, and multimeric conformations renders this inherently challenging. Moreover, structurally distinct serpin polymers are induced by chemical denaturants compared to pathological mutations or heating ([@bib10]). Techniques that can perturb equilibria by stabilizing particular structural endpoints (e.g., selective labeling, proteolysis, and/or peptide binding) may also skew characterization of intermediate ensembles in conformational diseases. We have therefore undertaken the residue-specific study of a *forme fruste* disease mutant in solution, under physiological conditions, using minimally perturbing techniques. This is arguably the best strategy for studying intermediate states relevant to conformational diseases.

Lys154Asn α~1~-antitrypsin populates a polymerogenic intermediate state at 37°C. Polymers formed from Lys154 mutants recapitulate the pathognomic 2C1 neo-epitope. Polymerization of Lys154Asn α~1~-antitrypsin is therefore a good structural model for the pathological polymerization in vivo of more common and severe variants, such as Z α~1~-antitrypsin. Serendipitously, polymerization occurs slowly enough to allow the study of the solution behavior of α~1~-antitrypsin when the pathological intermediate state is populated. We have overcome obstacles posed by the molecule\'s large size (45 kDa) to use NMR spectroscopy and characterize residues that remain native-like or alter their solution behavior in association with formation of the pathological intermediate. Our data are consistent with sufficient population of the intermediate state in Lys154Asn α~1~-antitrypsin at physiological temperatures to contribute to our observations. Most observed residues behave in a highly native-like way under these conditions. Relative intensity values indicate preservation of native-like signal after correcting for changes due to the mutation at 25°C and changes in protein tumbling and polymerization at 37°C. A value of 100% would therefore indicate entirely native-like structural and dynamic behavior in a particular residue. The mean relative intensity across the entire subset of residues that could be analyzed in this way was 82%. It is therefore unlikely that the intermediate ensemble is substantially unfolded as required for extensively domain-swapped models of polymerization. Specifically, analysis of cross-peak intensities supports high stability in the hI-s5A region proposed to unfold in the β-hairpin model ([Figure 3](#fig3){ref-type="fig"}C). This is indicated by relative intensities of 90% for the seven residues within this region compared with the equivalent cross-peaks in the native state. Similar preservation of native-like signal is indicated for the core strands 4 and 5 of β sheet B (residues 370--388).

Larger changes ([Figures 3](#fig3){ref-type="fig"}A and 3B) may be due to a number of differences in structural and/or dynamic behavior, so their interpretation is complex. Nevertheless, the localization of the greatest changes from wild-type native behavior is striking. Such changes are reported by residues in regions associated with remodeling during formation of the intermediate state in parsimonious models of polymerization ([Figure 3](#fig3){ref-type="fig"}B, left panel, circled: Thr165 at the top of hF, relative intensity 0.46, Phe189, upper s4a, 0.31). The effects on these residues cannot be simply explained by proximity to the mutation site, since they lie further from it than many residues that report highly native-like behavior. Residues reporting a major loss of relative intensities are also observed at the C-terminal end of hA ([Figure 3](#fig3){ref-type="fig"}B, right panel ellipse: Arg39, relative intensity 0.11 and His 43, 0.33), even further from the mutation site. This corresponds to the site of another polymerogenic α~1~-antitrypsin deficiency mutation (Ile39Cys; [@bib26]). Mutations in nearby serpin residues (Tyr38Ser, antithrombin; Leu41Pro, α~1~-antitrypsin) are also associated with deficiency and disease ([@bib5]). Our data suggest that structural and/or dynamic changes here are associated with formation of the pathological intermediate. The effects would be transmitted most directly to the A-helix from s3A via the intervening shutter and s6B motif ([Figure 3](#fig3){ref-type="fig"}D), known hotspots for pathological, polymerogenic mutations.

Taking together the data from biochemical, biophysical, enzyme-linked immunosorbent assay (ELISA) and cell studies, and previous observations ([@bib3; @bib38]), we propose that the helix F and linker region constitutes a "clasp" motif ([Figure 3](#fig3){ref-type="fig"}D, purple). This may seal a network of interactions that regulate conformational change. Compromise of the network is associated with formation of the pathological intermediate.

Our data indicate that the methods used to induce intermediate formation when studying conformational diseases can be major determinants of the structural distribution of intermediate states within the solution ensemble. A recent study undertook residue-specific labeling of single Cys mutants during early guanidine denaturation. It supported extensive unfolding of strands 5 and 6, as well as helices F and I ([@bib22]). That pattern differs from our observations, both in the presence of the Lys154Asn mutation at 37°C and in low concentrations of urea. The modulation of monomeric ensembles to produce analogous but structurally distinct polymer states is relevant to studies of other conformational diseases, where chemical denaturants are commonly used in vitro to induce multimerization. It may also explain the phenomenon of strain specificity observed in conformational disorders, such as prion diseases and amyloidoses.

We have demonstrated that NMR spectroscopy is optimal for studying pathological intermediate formation in the serpinopathies, providing residue-specific detail without perturbing dynamic solution equilibria. Comparison of wild-type α~1~-antitrypsin characteristics with those of mild deficiency variants also powerfully dissects disease-relevant behavior. Such studies appear timely, given the range of proposed polymerisation models based upon data obtained using a range of more perturbing techniques. Further studies using different variants and NMR modalities, for example, detailed dynamics studies, will further characterize this contentious mechanism of pathological conformational change to optimize drug design and so treat the serpinopathies.

Experimental Procedures {#sec4}
=======================

Molecular Biology, Protein Purification, and Cell-Free Characterization {#sec4.1}
-----------------------------------------------------------------------

Mutations were introduced into α~1~-antitrypsin cDNA within the pQE31 and/or the pcDNA3.1 plasmids by polymerase chain reaction mutagenesis ([@bib42; @bib17]). pQE31 plasmids containing cDNA encoding hexahistidine-tagged, recombinant α~1~-antitrypsin were transfected into XL1 Blue *Escherichia coli* (Stratagene, Santa Clara, California, USA). The proteins were expressed and purified as previously described ([@bib31]). They were characterized using SDS-, nondenaturing and transverse urea gradient (TUG)-PAGE, circular dichroism (CD), intrinsic and 8-anilinonaphthalene-1-sulfonate (ANS) fluorescence spectrometry and by enzyme inhibitory activity and kinetics assays ([@bib6; @bib7; @bib20; @bib39]).

Ion Mobility Mass Spectrometry {#sec4.2}
------------------------------

Samples were buffer exchanged into 100 mM ammonium acetate (pH 7.0), desalted, and concentrated to a final concentration of 20 μM. Proteins were introduced to the mass spectrometer by nano electrospray ionization. All IM-MS experiments were performed in a hybrid quadrupole, orthogonal acceleration time-of-flight (oa-TOF) mass spectrometer equipped with a traveling wave (T-Wave) ion mobility separation device (Synapt HDMS, Waters, Manchester, UK; [@bib33]). The source temperature was 40°C, capillary voltage optimized 1.0--1.2 kV, and cone voltage was 20 V. The pressure in the T-Wave ion mobility cell was 0.55 mbar. The mobility gas was nitrogen. T-Wave height and velocity were set at 10 V and 300 m/s, respectively. Arrival time distributions were converted to collision cross-sections (CCS) by power fit ([@bib18; @bib40]). The calibrant protein was equine myoglobin (Sigma-Aldrich, Gillingham, UK). Data acquisition and processing were carried out using MassLynx 4.1 software (Waters Corp., Milford, MA, USA). Data are the mean (+/− SD) of three repeats.

Sample Preparation for NMR Spectroscopy {#sec4.3}
---------------------------------------

cDNA coding for hexahistidine-tagged α~1~-antitrypsin within a pQE31 plasmid was transformed into strain BL21-Gold (DE3) *E. coli* (Stratagene). Cells were cultured in M9 minimal media (in H~2~O or D~2~O) at 37°C with 1 g/l of ^15^NH~4~Cl (Spectra Stable Isotopes, Columbia, MD, USA) and 2 g/l of either glucose or ^13^C-glucose (Sigma-Aldrich) as the sole nitrogen and carbon sources, respectively. Following induction of protein expression cells were grown for 6 hr, rather than the 4 hr, period used with rich media. In all other respects protein expression and purification was carried out as described above. Samples were stored in 25 mM Na~2~HPO~4~ (pH 8.0), 50 mM NaCl, and 1 mM ethylenediaminetetraacetic acid. Sample homogeneity was confirmed by SDS-, nondenaturing and transverse urea gradient (TUG)-PAGE and by assessment of inhibitory activity.

NMR Spectroscopy Conditions {#sec4.4}
---------------------------

NMR spectra used to compare wild-type and Lys154Asn α~1~-antitrypsin were collected on uniformly ^15^N-labeled samples at 298 K, 175 μM on a Varian UnityInova 600 MHz spectrometer with an HCN cryoprobe. Ten percent D~2~O/1% 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) was added to concentrated α~1~-antitrypsin samples prior to NMR spectroscopy.

Backbone assignments were obtained from TROSY ([@bib32]) versions of HNCO, HN(CA)CO, HNCA, HN(CO)CA, HNCACB, and HN(CO)CACB spectra ([@bib11; @bib35]), together with a 3D NOESY-TROSY-^15^N HSQC spectrum ([@bib44]). ^2^H decoupling (1 kHz WALTZ-16; [@bib37]) was applied in triple-resonance experiments. Magnetization was transverse on CA or CB. Two-dimensional TROSY-^15^N HSQC spectra for assignment. Wild-type and Lys154Asn α~1~-antitrypsin comparison spectra were recorded using single-transition-to-single-transition polarization transfer and phase cycling for coherence order selection ([@bib34; @bib44]). Spectra were processed and analyzed using nmrPipe ([@bib8]) and CCPN ([@bib12]). All spectra were referenced to DSS at 0.0 ppm, manually phased and baseline corrected. Lys154Asn α~1~-antitrypsin spectra were analyzed by changes in cross-peak intensity relative to the data on wild-type α~1~-antitrypsin (I~K154N~/I~wild-type~) and magnitude of chemical shift change (Δδ). This was calculated as$$\sqrt{\left( {\Delta\delta^{1}\text{H}} \right)^{2} + \left( {\Delta\delta^{15}\text{N}/5} \right)^{2}},$$where Δ^1^H was the change in the chemical shift along the ^1^H axis andΔ^15^N the change in chemical shift along the ^15^N axis (parts per million \[ppm\]).

Cell Biological Characterization {#sec4.5}
--------------------------------

The pcDNA3.1 plasmids containing wild-type and mutant α~1~-antitrypsins were transiently transfected into COS-7 cells. The resulting protein expression was characterized by western blot analyses of SDS- and nondenaturing PAGE of intracellular material as previously described ([@bib28; @bib27]). Luciferase controls were used to confirm equivalent loading between samples. The polymer load was quantified by ELISA using the polymer-specific 2C1 monoclonal antibody as the primary antibody ([@bib29]).

Supplemental Information {#app2}
========================

Document S1. Figures S1--S3

The authors wish to thank Dr. James Irving (Department of Medicine, Cambridge Institute for Medical Research, University of Cambridge, UK) for helpful discussions during the preparation of this paper. M.P.N. received a Wellcome Trust Value in People Award; G.L. and A.O.M.P. hold Wellcome Trust programme PhD Studentships; U.I.E. is an MRC Clinical Training Fellow; and B.G. is a Wellcome Trust Intermediate Clinical Fellow. This work was supported by funding from the Wellcome Trust, the Medical Research Council (UK), the Human Frontier Science Programme, the Biotechnology and Biological Sciences Research Council, Papworth Hospital NHS Trust, and the Birkbeck College Faculty Research Fund.

Supplemental Information includes three figures and can be found with this article online at [doi:10.1016/j.str.2012.01.012](10.1016/j.str.2012.01.012).

![Pathological Polymerization of Lys154Asn α~1~-Antitrypsin\
(A) Polymerisation pathway from native conformer (mutation site circled).\
(B) 7.5% (w/v) native PAGE; polymerization of wild-type and Lys154Asn α~1~-antitrypsin (0.5 mg/ml protein \[pH 7.4\]) in vitro over 12 days at 37°C and 42°C. Polymerization is reported by loss of the monomeric band and the appearance of aggregated protein. (Right) Polymers formed by 30 min incubation between 40°C and 50°C of nonglycosylated wild-type (blue) and Lys154Asn (red) α~1~-antitrypsin are detected by 2C1 mAb ELISA (Z α~1~-antitrypsin polymer calibrated; [@bib29]). Differences between wild-type and mutant are significant (p \< 0.05 to p \< 0.005) for all temperatures. The increase in signal relative to starting material reaches significance for Lys154Asn α~1~-antitrypsin for incubations at 47.5 (p \< 0.05) and 50 (p \< 0.005)°C. Data are mean ± standard deviation (error bars) of three experiments.\
(C) (Left) Thermal denaturation CD spectroscopy (left; mean ellipticity at 222 nm, n = 10) for wild-type (blue) and Lys154Asn (red) α~1~-antitrypsin. (Right) Arrival times and collision cross-section (CCS) values calculated by IM-MS for wild-type (blue) and Lys154Asn α~1~-antitrypsin indicated a 7.8% increase in CCS in Lys154Asn relative to wild-type α~1~-antitrypsin at 39°C (no difference at 20°C and 34°C). Data are mean ± standard deviation (error bars) of three experiments.\
See also [Figure S1](#app2){ref-type="sec"}.](gr1){#fig1}

![^1^H-^15^N TROSY-HSQC Spectrum of Wild-Type and Lys154Asn α~1~-Antitrypsin\
Zoom (right) illustrates examples of reporter residues (wild-type, black; Lys154Asn, red).\
See also [Figure S2](#app2){ref-type="sec"}.](gr2){#fig2}

![Population of the Intermediate by Lys154Asn α~1~-Antitrypsin under Physiological Conditions\
(A) Differential change in intensity for reporter cross-peaks in the ^1^H-^15^N TROSY-HSQC of Lys154Asn α~1~-antitrypsin at 37°C. Intensities are scaled relative to the wild-type protein at the same temperature and the intensities observed for Lys154Asn α~1~-antitrypsin relative to the wild-type at 25°C \[(I~K154N,37~/I~wt,37~)/(I~K154N,25~/I~wt,25~)\]. All intensities were scaled for concentration and 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) control intensity. Error bars are defined according to variability observed in the datasets from which the values are derived, according to the formula$$\left\lbrack {\left. {\left( \frac{\text{I}_{\text{K}154\text{N},37}}{\text{I}_{\text{WT},37}} \right)/\left( \frac{\text{I}_{\text{K}154\text{N},25}}{\text{I}_{\text{WT},25}} \right)} \right\rbrack \times \sqrt{\left( \frac{\text{d}_{\text{K}154\text{N},37}}{\text{I}_{\text{K}154\text{N},37}} \right)^{2} + \left( \frac{\text{d}_{\text{WT},37}}{\text{I}_{\text{WT},37}} \right)^{2} + \left( \frac{\text{d}_{\text{K}154\text{N},25}}{\text{I}_{\text{K}154\text{N},25}} \right)^{2} + \left( \frac{\text{d}_{\text{WT},25}}{\text{I}_{\text{WT},25}} \right)^{2}}} \right.,$$where I is the intensity for data obtained from wild-type (WT) or Lys154Asn (K154N) α~1~-antitrypsin at 25° or 37°C as indicated by the subscripts and d terms represent the observed standard deviation of peak intensities within each spectra.\
(B) Relative intensities quantified in (A) mapped onto the subset of reporter residues (spheres) in red-white-blue heatmap coloring (increases, red; unchanged, white; reductions, blue). Greatest change (maximally blue by RGB, residue 39) corresponds to a relative intensity of 0.11. Increasing redness indicates increasing intensity on the same scale. Ellipse: hA region reporting major change from native-like intensity.\
(C) (Left) Intensity change and stability observed in β sheet A, hF, hI, and connecting linkers. Current structural models of polymerogenic intermediates in the parsimonious (center) and β-hairpin (right) linkage models of polymerisation for comparison (β strand 5A magenta; hI and connecting linker cyan).\
(D) (Left) Unlocking the clasp. Proposed scheme of polymerogenic intermediate formation through propagation of conformational change from the mutation site through β sheet A (blue) and more distant motifs. The Lys154Asn mutation abolishes a hydrogen bond between hF and the neighboring linker (clasp, purple) region (I). Under physiological conditions this destabilization causes remodeling of the upper part of the clasp (II). Consequent structural changes in the upper part of s3A (III) facilitate lateral movement required to open the s4A site. Changes in s3A are also transmitted via (IV) the shutter region and s6B (brown) to the C-terminal residues of hA (V). Most of the residues within β sheet A remain within a native-like environment during formation of the intermediate. This is seen not only for residues in s5A but also for residues in hI and the connecting linker. (Right) Factors stabilizing (blue, arrow denotes stabilizing displacement of clasp region induced by the Gly117Phe mutation; [@bib17]) and destabilizing (red) the network of regulatory interactions.\
See also [Figure S3](#app2){ref-type="sec"}.](gr3){#fig3}

[^1]: These authors contributed equally

[^2]: These authors contributed equally