Urbina 2018 — Biomining dissertation: isothermal-titration characterisation of natural and rationally-designed Cu/Zn/Ni-binding peptides, and a flagellin-knockout strain for surface-display metal recovery

Urbina’s PhD dissertation at UC Santa Cruz frames biomining (microbial extraction of metals from materials that are not traditionally mined, principally end-of-life electronics) as the application context and then reports two pieces of primary work in support of that goal. Chapter 1 is a review of the state of biomining (biooxidation of metal sulfides, heap bioleaching, basalt and silicate-mineral bioleaching, electronic-component bioleaching, and bioengineering of bacterial surfaces). Chapter 2 measures the intrinsic binding affinity (Kₐ, ΔH, n) of eight short peptides for Cu²⁺, Zn²⁺, and Ni²⁺ by isothermal titration calorimetry (ITC) and the apparent affinity for Cu when a competing ion (Zn or Ni) is pre-bound, contrasting naturally-occurring metal-binding motifs against motifs designed in silico by Kožíšek et al. (2008). Chapter 3 constructs and characterises a Bacillus subtilis flagellin-knockout strain (PY79 ΔhagAB::ery) intended as a chassis for displaying random peptide libraries on flagella for affinity-based screening of metal-binding peptides. The dissertation does not measure heavy metals in food, cosmetics, or any human-exposure matrix; its relevance to the Heavy Metal Index is to the peptide-Cu / peptide-Ni / peptide-Zn coordination-chemistry literature that already includes luo2024-peptides-heavy-metal-remediation, shalev2022-peptide-metal-nmr-review, spallacci2025-bioinformatics-biomimetic-metal-peptide, zhang2025-metalorian-de-novo-metal-binding-peptides, and grill1989-phytochelatins-heavy-metal-binding-peptides-plants.

Why this matters

• It is the first dissertation in the wiki’s peptide-metal-binding corpus to put naturally-occurring metal-binding motifs and computationally-designed motifs through the same ITC pipeline under matched conditions (10 mM MES buffer, pH 5.5, 25 °C, Malvern MicroCal iTC200, Origin-7 one-site model). This makes the resulting Kₐ values directly comparable in a way that the published-literature affinity values they reference (collected across different buffers, pH values, and instruments) are not.
• The two natural HypB-derived motifs (HypB1 CTTCGCG, HypB2 MCTTCGCGEG) and the rationally-designed HHTC and CHSK peptides all bind Cu²⁺ with Kₐ in the 10⁶ M⁻¹ range. The consensus Cu-binding motif Cu-02 (HCWCHM) is the highest-affinity peptide at Kₐ ≈ 9.9 × 10⁶ M⁻¹.
• The Zn-targeted rational-design peptides KDTK and KDKD bind Cu more strongly than they bind Zn (consistent with the Irving–Williams stability series Mg²⁺ < Mn²⁺ < Fe²⁺ < Co²⁺ < Ni²⁺ < Cu²⁺ > Zn²⁺). The dissertation reports this as a limit on rational design when the target metal sits below Cu in the series.
• The competition data establish that for the HHTC and CHSK rationally-designed peptides, Cu affinity is lowered by an order of magnitude when Zn or Ni is pre-bound; for the consensus Cu-02 peptide, Cu affinity is lost entirely when Ni is the competing ion. The natural HypB2 motif retains Cu affinity in the presence of both Zn and Ni.
• The exposure-context paragraph in Chapter 1 cites Shantou University Medical College finding 82 % of resident children near the Guiyu, China e-waste processing site have lead poisoning suspected to be caused by e-waste contamination. This is the single human-exposure datapoint in the dissertation; it is a citation to other work, not a measurement reported here.

Key numbers

Peptides assessed (Chapter 2, Table 1, p. 38).

Intrinsic and apparent association constants Kₐ (Chapter 2, Table 2, p. 39; supporting text pp. 28-32). “Zn → Cu” denotes the apparent Kₐ for Cu when Zn is already in solution as a competing ion (titrated as Cu into the pre-formed peptide-Zn complex); “Ni → Cu” likewise.

† Paper-internal discrepancy: Table 2 (p. 39) reports KDTK Zn Kₐ = (2.44 ± 3.53) × 10⁴ M⁻¹, whereas the body text on p. 32 reports “Kₐ = (2.44 ± 3.53) M⁻¹ for Zn.” Both values are recorded here; the table value is more consistent with the dissertation’s own framing of KDTK as a Zn-designed peptide that nonetheless binds Cu with at-least-comparable affinity. A reader who needs to use this value should consult the supplementary raw ITC traces.

‡ Paper-internal discrepancy: Table 2 (p. 39) reports KDKD Cu Kₐ = (1.27 ± 0.11) × 10⁴ M⁻¹, whereas the body text on p. 32 reports “(1.27 ± 1.13) × 10⁴ M⁻¹.” The central value matches; the uncertainty differs by an order of magnitude. The table value (smaller error bar) is recorded above for consistency with how KDTK is handled.

Published-literature Cu Kₐ value cited as comparator for the HHTC peptide (Chapter 2, text on pp. 28-29).

Truncation / replacement series for the KDETSY residues hypothesised to interact with competing metal ions (Chapter 2, Table 3 region, p. 31-32).

Heat-of-dilution / mixing background reference values (Methods, p. 25).

Buffer ionisation enthalpies relevant to the pH-cross-comparison (Discussion, p. 29).

Speciation modelling parameters (Methods, p. 24-25; Discussion, p. 29).

Cited exposure-context datapoint (Chapter 1, Introduction, p. 1).

Methods (brief)

Peptide synthesis. Eight peptides synthesised by Elim Biopharmaceuticals (Hayward, CA), HPLC-purified with HCl as the counter-ion, lyophilised, purity >98 % verified by mass spectrometry. N-terminal acetylation and C-terminal amidation were applied to neutralise terminal charge. Peptide concentration determined by Pierce™ BCA Assay.

Isothermal titration calorimetry. Malvern MicroCal iTC200 calorimeter (NASA Ames Space Biosciences Division, Mountain View, CA). 25 °C; 20 injections at 0.5–1 µL each; 150-second injection intervals. Buffer: 10 mM 2-(N-morpholino)-ethanesulfonic acid (MES) at pH 5.5 (the dissertation reports MES was chosen because it does not cause metal-ion interference via complexation or amine oxidation, is stable over pH 3–11, and has a stable pKa across 15–45 °C; citations Wang and Lawrence 1989; Kandegedara and Rorabacher 1999). Peptide concentration typically 0.5 mM in the cell; metal-chloride salt concentrations 2.4–6.4 mM in the syringe (10-20× excess over peptide). For low-affinity titrations, syringe concentrations up to 100× the cell were used. Metal chlorides: CuCl₂·2H₂O (Baker Analyzed ACS), NiCl₂, ZnCl₂. Reference titrations for instrument validation: Ca–EDTA kit (Malvern Panalytical), all parameters within manufacturer specification. Raw data corrected for heat of dilution by subtracting the average of three blank titrations (metal-into-buffer, buffer-into-peptide, buffer-into-buffer). Integrated heat data fit with a one-site binding model in Origin-7 (MicroCal). Heat of ionisation of MES was determined to be negligible (Freyer and Lewis 2008) and no correction was applied.

ICP-OES. Metal stock concentrations verified on a Thermo iCAP 7400 ICP-OES at the UC Santa Cruz Marine Analytical Laboratory.

Speciation modelling. Visual MINTEQ 3.0 (Gustafsson 2007) used to verify metal-ion speciation under the experimental conditions. Outputs included in the dissertation’s supplementary materials.

Competition assays. For each peptide, either (a) a competing ion (Zn²⁺ or Ni²⁺) was titrated into a pre-formed peptide–Cu complex, or (b) Cu²⁺ was titrated into a pre-formed peptide–Zn or peptide–Ni complex. The latter set yields the “Zn → Cu Kₐ” and “Ni → Cu Kₐ” apparent constants tabulated above.

Flagellar-display strain construction (Chapter 3). Bacillus subtilis PY79 was the starting strain. The hag locus (encoding flagellin) was disrupted by allelic-replacement using an erythromycin-resistance cassette to yield the ΔhagAB::ery strain. Flagellin variants were re-introduced via integration vectors. Adsorption screens were performed against Au(III) and other electronic-waste-relevant metals; chapter results are reported as figures, with replicate-level numerical data in the chapter’s figures section (pp. 60-65).

What this dissertation does not measure. No dietary, cosmetic, drinking-water, or human-exposure concentrations. No food matrix, no contaminant occurrence values, no regulatory threshold work. The 82 % Pb-poisoning prevalence in Guiyu children quoted in the introduction is a citation to Shantou University Medical College, not a measurement reported by this dissertation.

Implications

Certification: Not directly applicable. Copper, nickel, and zinc are all in the HMI metal taxonomy (copper, nickel, zinc); Ni is on the HMTc-certified analyte list (Pb, tAs, Cd, MeHg, tHg, iAs, Ni, Al, Cr-VI, Sn), Cu and Zn are not. The dissertation measures no food or supply-chain matrix and therefore cannot contribute occurrence data to any product-category page.

Courses: Marginal. Useful as worked example in a future advanced module on peptide-based metal chelation chemistry — specifically, how ITC characterises peptide-metal affinity, how the Irving–Williams series constrains rational design when the target metal sits below Cu, and how competing ions can lower apparent affinity for the cognate metal without producing a measurable isotherm for the competitor.

App: Not applicable. No contamination-profile data.

Microbiome: Not applicable. No microbiota or microbial-community measurements; Chapter 3’s Bacillus subtilis work uses the organism as an engineered chassis, not as a human-microbiome species under environmental stress.

Verification notes

• Filename / content mismatch. The PDF is filed under the Kimi folder as 44_Functional_Interplay_Between_the_Hippo_Pathway_and_Heavy_Met.pdf, but the actual contents are the Urbina (2018) UC Santa Cruz dissertation on biomining and peptide-metal binding. There is no Hippo-pathway content in this file. The raw_handle has been preserved per the manual-fetch convention (MFK_ handle follows the file as Kimi delivered it); the cite_key reflects the actual content. A future session that needs the actual Hippo-pathway / heavy-metals paper will need to fetch it separately; this PDF does not contain it.
• DOI / access_url fallback. UCSC dissertations are typically deposited in eScholarship (UC’s open-access repository); no_doi_assigned: true and the access_url points at the UCSC ETD collection landing page rather than the specific item, because the exact item URL was not verified during ingest. Both should be backfilled (item-level URL, and DOI if eScholarship has assigned one) when verified.
• Paper-internal numerical discrepancies (three). The dissertation contains three independent body-text-vs-Table-2 disagreements that are footnoted above where they appear: (1) KDTK Zn Kₐ — body p. 32 reads “(2.44 ± 3.53) M⁻¹” vs Table 2 p. 39 “(2.44 ± 3.53) × 10⁴ M⁻¹”; (2) KDKD Cu Kₐ — body p. 32 reads “(1.27 ± 1.13) × 10⁴” vs Table 2 “(1.27 ± 0.11) × 10⁴” (central value matches; uncertainty differs by 10×); (3) HHTC intrinsic Cu Kₐ — body p. 28 reads “(1.89 ± 0.3) × 10⁶” vs Table 2 “(1.74 ± 0.49) × 10⁶”. In all three cases the Table 2 value is used in the intrinsic-Kₐ table above and the body-text value is footnoted; the table is treated as the canonical record because it carries the dissertation’s full uncertainty bookkeeping.
• Audit subagent (2026-06-08) flagged misattribution of the (2.4 ± 0.5) × 10⁶ Cu Kₐ comparator. Initial draft labelled this value as the HypB1 comparator; verified against PDF p. 28–29 — the value is the published HHTC comparator from Kožíšek et al. 2008, not a HypB1 value. The published-literature comparator table has been corrected and the HypB1 row removed.
• Audit subagent (2026-06-08) flagged statistic descriptor. The HHTC-Tr P-value is from an unpaired (not just “two-tailed”) t-test per p. 31; descriptor updated to “unpaired t-test … two-tailed P”.
• Audit subagent (2026-06-08) flagged BCA-assay attribution. Initial draft wrote “(Thermo Fisher)” after Pierce BCA; PDF p. 23 reads only “Pierce™ BCA Assay” without Thermo Fisher attribution (Pierce is a Thermo brand, but adding it is wiki-added context). Reverted to source wording.
• Audit subagent (2026-06-08) flagged MES buffer rationale. Initial draft paraphrased the MES choice as “to keep histidine pKa ≈ 6 within range” — that specific framing is wiki-added interpretation. Rewritten to use the dissertation’s own three-criterion rationale (no metal-ion interference via complexation or amine oxidation; stable over pH 3–11; stable pKa across 15–45 °C).
• Evidence tier C. Assigned because this is a PhD dissertation, not peer-reviewed at journal-article level; the binding affinity data are internally consistent and reference-method (ITC) but have not been re-published in a peer-reviewed venue (no DOI located).

Wiki pages updated on ingest

• copper
• nickel
• zinc

Page history

The five most recent substantive edits to this page. The full version history lives in git; when DOI minting comes online (see schema docs), each entry below will also link to a version-pinned DataCite DOI.