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1. From peptides to proteins: coiled-coil tetramers to single-chain 4-helix bundles

   https://doi.org/10.1039/d2sc04479j  

   Naudin, Elise A.; Albanese, Katherine I.; Smith, Abigail J.; Mylemans, Bram; Baker, Emily G.; Weiner, Orion D.; Andrews, David M.; Tigue, Natalie; Savery, Nigel J.; Woolfson, Derek N. (October 2022, Chemical Science)

   The design of completely synthetic proteins from first principles— de novo protein design—is challenging. This is because, despite recent advances in computational protein–structure prediction and design, we do not understand fully the sequence-to-structure relationships for protein folding, assembly, and stabilization. Antiparallel 4-helix bundles are amongst the most studied scaffolds for de novo protein design. We set out to re-examine this target, and to determine clear sequence-to-structure relationships, or design rules, for the structure. Our aim was to determine a common and robust sequence background for designing multiple de novo 4-helix bundles. In turn, this could be used in chemical and synthetic biology to direct protein–protein interactions and as scaffolds for functional protein design. Our approach starts by analyzing known antiparallel 4-helix coiled-coil structures to deduce design rules. In terms of the heptad repeat, abcdefg — i.e. , the sequence signature of many helical bundles—the key features that we identify are: a = Leu, d = Ile, e = Ala, g = Gln, and the use of complementary charged residues at b and c. Next, we implement these rules in the rational design of synthetic peptides to form antiparallel homo- and heterotetramers. Finally, we use the sequence of the homotetramer to derive in one step a single-chain 4-helix-bundle protein for recombinant production in E. coli . All of the assembled designs are confirmed in aqueous solution using biophysical methods, and ultimately by determining high-resolution X-ray crystal structures. Our route from peptides to proteins provides an understanding of the role of each residue in each design. 

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2. mebipred : identifying metal-binding potential in protein sequence

   https://doi.org/10.1093/bioinformatics/btac358  

   Aptekmann, A. A.; Buongiorno, J.; Giovannelli, D.; Glamoclija, M.; Ferreiro, D. U.; Bromberg, Y.; Cowen, ed., Lenore (May 2022, Bioinformatics)

   Abstract Motivationmetal-binding proteins have a central role in maintaining life processes. Nearly one-third of known protein structures contain metal ions that are used for a variety of needs, such as catalysis, DNA/RNA binding, protein structure stability, etc. Identifying metal-binding proteins is thus crucial for understanding the mechanisms of cellular activity. However, experimental annotation of protein metal-binding potential is severely lacking, while computational techniques are often imprecise and of limited applicability. Resultswe developed a novel machine learning-based method, mebipred, for identifying metal-binding proteins from sequence-derived features. This method is over 80% accurate in recognizing proteins that bind metal ion-containing ligands; the specific identity of 11 ubiquitously present metal ions can also be annotated. mebipred is reference-free, i.e. no sequence alignments are involved, and is thus faster than alignment-based methods; it is also more accurate than other sequence-based prediction methods. Additionally, mebipred can identify protein metal-binding capabilities from short sequence stretches, e.g. translated sequencing reads, and, thus, may be useful for the annotation of metal requirements of metagenomic samples. We performed an analysis of available microbiome data and found that ocean, hot spring sediments and soil microbiomes use a more diverse set of metals than human host-related ones. For human microbiomes, physiological conditions explain the observed metal preferences. Similarly, subtle changes in ocean sample ion concentration affect the abundance of relevant metal-binding proteins. These results highlight mebipred’s utility in analyzing microbiome metal requirements. Availability and implementationmebipred is available as a web server at services.bromberglab.org/mebipred and as a standalone package at https://pypi.org/project/mymetal/. Supplementary informationSupplementary data are available at Bioinformatics online. 

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3. Probing the Ni ²⁺ ‐selective Response of Fluorescent Probe NiSensor‐1 with the NiCast Photocaged Complex ^{† ‡}

   https://doi.org/10.1111/php.13567  

   Hickey, Erin E.; Kennedy, Daniel P.; Gwizdala, Celina; Basa, Prem N.; Müller, Peter; MacDonald, John; Burdette, Shawn C. (December 2021, Photochemistry and Photobiology)

   Abstract CTEA (N,N‐bis[2‐(carboxylmethyl)thioethyl]amine) is a mixed donor ligand that has been incorporated into multiple fluorescent sensors such as NiSensor‐1 that was reported to be selective for Ni²⁺. Other metal ions such as Zn²⁺do not produce an emission response in aqueous solution. To investigate the coordination chemistry and selectivity of this receptor, we prepared NiCast, a photocage containing the CTEA receptor. Cast photocages undergo a photoreaction that decreases electron density on a metal‐bound aniline nitrogen atom, which shifts the binding equilibrium toward unbound metal ion. The unique selectivity of CTEA was examined by measuring the binding affinity of NiCast and the CTEA receptor for Ni²⁺, Zn²⁺, Cd²⁺and Cu²⁺under different conditions. In aqueous solution, Ni²⁺binds more strongly to the aniline nitrogen atom than Cd²⁺; however, in CH₃CN, the change in affinity virtually disappears. The crystal structure of [Cu(CTEA)], which exhibits a Jahn–Teller–distorted square pyramidal structure, was also analyzed to gain more insight into the underlying coordination chemistry. These studies suggest that the fluorescence selectivity of NiSensor‐1 in aqueous solution is due to a stronger interaction between the aniline nitrogen atom and Ni²⁺compared to other divalent metal ions except Cu²⁺. 

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4. Zn-Enhanced Asp-Rich Antimicrobial Peptides: N-Terminal Coordination by Zn(II) and Cu(II), Which Distinguishes Cu(II) Binding to Different Peptides

   https://doi.org/10.3390/ijms22136971  

   Miller, Adriana; Matera-Witkiewicz, Agnieszka; Mikołajczyk, Aleksandra; Wątły, Joanna; Wilcox, Dean; Witkowska, Danuta; Rowińska-Żyrek, Magdalena (July 2021, International Journal of Molecular Sciences)

   null (Ed.)

   The antimicrobial activity of surfactant-associated anionic peptides (SAAPs), which are isolated from the ovine pulmonary surfactant and are selective against the ovine pathogen Mannheimia haemolytica, is strongly enhanced in the presence of Zn(II) ions. Both calorimetry and ITC measurements show that the unique Asp-only peptide SAAP3 (DDDDDDD) and its analogs SAAP2 (GDDDDDD) and SAAP6 (GADDDDD) have a similar micromolar affinity for Zn(II), which binds to the N-terminal amine and Asp carboxylates in a net entropically-driven process. All three peptides also bind Cu(II) with a net entropically-driven process but with higher affinity than they bind Zn(II) and coordination that involves the N-terminal amine and deprotonated amides as the pH increases. The parent SAAP3 binds Cu(II) with the highest affinity; however, as shown with potentiometry and absorption, CD and EPR spectroscopy, Asp residues in the first and/or second positions distinguish Cu(II) binding to SAAP3 and SAAP2 from their binding to SAAP6, decreasing the Cu(II) Lewis acidity and suppressing its square planar amide coordination by two pH units. We also show that these metal ions do not stabilize a membrane disrupting ability nor do they induce the antimicrobial activity of these peptides against a panel of human pathogens. 

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5. Emerging investigator series: characterization of silver and silver nanoparticle interactions with zinc finger peptides

   https://doi.org/10.1039/C9EN00065H  

   Park, Grace; Amaris, Zoe N.; Eiken, Madeline K.; Baumgartner, Karl V.; Johnston, Kathryn A.; Williams, Mari A.; Marckwordt, Jasmine G.; Millstone, Jill E.; Splan, Kathryn E.; Wheeler, Korin E. (January 2019, Environmental Science: Nano)

   In biological systems, chemical and physical transformations of engineered silver nanomaterials (AgENMs) are mediated, in part, by proteins and other biomolecules. Metalloprotein interactions with AgENMs are also central in understanding toxicity, antimicrobial, and resistance mechanisms. Despite their readily available thiolate and amine ligands, zinc finger (ZF) peptides have thus far escaped study in reaction with AgENMs and their Ag( i ) oxidative dissolution product. We report spectroscopic studies that characterize AgENM and Ag( i ) interactions with two ZF peptides that differ in sequence, but not in metal binding ligands: the ZF consensus peptide CP-CCHC and the C-terminal zinc finger domain of HIV-1 nucleocapsid protein p7 (NCp7_C). Both ZF peptides catalyze AgENM (10 and 40 nm, citrate coated) dissolution and agglomeration, two important AgENM transformations that impact bioreactivity. AgENMs and their oxidative dissolution product, Ag( i )(aq), mediate changes to ZF peptide structure and metalation as well. Spectroscopic titrations of Ag( i ) into apo-ZF peptides show an Ag( i )–thiolate charge transfer band, indicative of Ag( i )–ZF binding. Fluorescence studies of the Zn( ii )–NCp_7 complex indicate that the Ag( i ) also effectively competes with the Zn( ii ) to drive Zn( ii ) displacement from the ZFs. Upon interaction with AgENMs, Zn( ii ) bound ZF peptides show a secondary structural change in circular dichroism spectroscopy toward an apo-like structure. The results suggest that Ag( i ) and AgENMs may alter ZF protein function within the cell. 

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