CBP Family α — ATCUN baseline library

This document describes Family α of the CBP peptide library: the minimal-confounder ATCUN baselines designed for Cu(II) capture in water, with no internal HxxxH clamp and a controlled C-terminal anchor (where present).

Family α peptides are the reference set used to quantify how much Cu(II) sequestration alone can shift Aβ conformational/aggregation metrics (including any detectable increase in α-helical population).

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1) What “Family α” means (operational definition)

A peptide belongs to Family α if it satisfies all of the following:

• ATCUN-like head present: N-terminus starts with DAH or GGH (H₂N–X–X–His)
• N-terminus is free (not acetylated) so the ATCUN-like Cu(II) binding mode is enabled
• No internal HxxxH (i,i+4) clamp beyond the head region
• Helical, soluble scaffold (EAAK-type repeats)
• Optional single Trp-based anchor at the C-terminus (LWKK, AWKK, or WKK)

Design intent: maximize interpretability (clean metal binding + helix scaffold) while minimizing additional interaction levers.

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2) Family α members (ordered by length)

Source of truth: CBP_mapping_enriched.csv

• CBPα1: GGHEAAKEAAKEAAKLWKK (19 aa)
  Role: ATCUN-variant head (GGH) to probe sensitivity to the exact ATCUN-like sequence.

• CBPα2: DAHEAAKEAAKEAAKEALWKK (21 aa)
  Role: Baseline ATCUN (DAH) with a moderate LWKK anchor.

• CBPα3: DAHEAAKEAAKEAAKEAAKWKK (22 aa)
  Role: Minimal anchor (WKK), reduced hydrophobicity vs LWKK.

• CBPα4: DAHEAAKEAAKEAAKEAAKAWKK (23 aa)
  Role: Softer anchor (A-WKK)—a mild reduction in hydrophobic engagement.

• CBPα5: DAHEAAKEAAKEAAKEAAKEAAKEAAKEAAKLWKK (35 aa)
  Role: Long baseline scaffold (stronger intrinsic helicity from length).

• CBPα6: GGHEAAKEAAKEAAKEAAKEAAKEAAKEAAKLWKK (35 aa)
  Role: Long baseline with GGH head; isolates head-effect at fixed length.

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3) Why Family α exists (scientific rationale)

3.1 ATCUN-like Cu(II) capture with minimal ambiguity

ATCUN-like motifs (H₂N–X–X–His) typically bind Cu(II) strongly via a defined N-donor set involving:

• free N-terminal amine
• backbone amide nitrogens
• His(3) imidazole

Family α uses this as the primary functional lever.

3.2 EAAK scaffolding for solubility and helical conditioning

EAAK-like repeats:

• increase aqueous solubility,
• stabilize α-helical propensity (especially for ≥30 aa),
• reduce aggregation driven by hydrophobic segments.

3.3 Single Trp anchor (optional) as a controlled interaction handle

When present, a single Trp-based tail (…WKK) aims to provide weak hydrophobic engagement (e.g., toward Aβ hydrophobic patches) without introducing β-zipper-prone motifs.

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4) Practical caveats (critical for correct interpretation)

4.1 Free N-terminus is mandatory

ATCUN-like binding assumes a free N-terminus.
Do not acetylate α-family peptides; if acetylated, they belong to γ controls by design.

4.2 pH and ionic strength dependence

Cu(II) capture and any peptide–Aβ interaction are sensitive to:

• pH (His protonation; backbone amide deprotonation contribution)
• ionic strength (electrostatic screening)

Always report exact conditions.

4.3 “Cu sequestration” vs “α induction” are distinct outcomes

Family α is optimized to answer “does Cu removal alone help?”
If you observe α-helicity changes in Aβ, validate that:

• it is not an artifact of concentration/ionic strength,
• it scales with Cu occupancy on the peptide.

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5) Recommended readouts (Family α baseline workflow)

For each CBPα peptide:

5.1 Apo peptide

• Helicity (DSSP or CD proxy)
• Self-association check (contacts / aggregation propensity)

5.2 Cu(II)-bound peptide

• Cu coordination stability (Cu–N distances, coordination number, geometry)
• Cu exposure (SASA around Cu)

5.3 Competition system (Aβ + Cu + CBPα)

• Fraction of Cu bound to peptide vs Aβ (dominant occupancy)
• Aβ helicity / secondary-structure shift (DSSP / CD-deconvolution strategy)
• Aβ–peptide contacts (residence times, hotspots)
• Aβ aggregation-prone metrics (β-content, inter-Aβ contacts, compaction proxies)

If feasible:

• ΔG_exchange for Aβ–Cu + CBPα → Aβ + CBPα–Cu

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6) How to use Family α in decision-making

Baseline ranking rule (within α): Prefer peptides that:

1. dominate Cu occupancy in competition,
2. remain helical/soluble without self-association,
3. minimize non-specific binding while still enabling measurable Aβ modulation.

Interpretation rule: Any improvement observed with β/δ families must be reported relative to α baselines to quantify the incremental value of clamps or increased electrostatics.

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