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1. Salt bridges govern the structural heterogeneity of heme protein interactions and porphyrin networks: microperoxidase-11

   https://doi.org/10.1039/d0ra04956e  

   Porter, J. ; Dit Fouque, K. Jeanne ; Miksovska, J. ; Fernandez-Lima, F. ( September 2020 , RSC Advances)

   null (Ed.)

   In this work, a proteolytic digest of cytochrome c (microperoxidase 11, MP-11) was used as a model to study the structural aspects of heme protein interactions and porphyrin networks. The MP-11 structural heterogeneity was studied as a function of the starting pH ( e.g. , pH 3.1–6.1) and concentration ( e.g. , 1–50 μM) conditions and adduct coordination. Trapped ion mobility spectrometry coupled to mass spectrometry (TIMS-MS) showed the MP-11 structural dependence of the charge state distribution and molecular ion forms with the starting pH conditions. The singly charged ( e.g. , [M] + , [M − 2H + NH 4 ] + , [M − H + Na] + and [M − H + K] + ) and doubly charged ( e.g. , [M + H] 2+ , [M − H + NH 4 ] 2+ , [M + Na] 2+ and [M + K] 2+ ) molecular ion forms were observed for all solvent conditions, although the structural heterogeneity ( e.g. , number of mobility bands) significantly varied with the pH value and ion form. The MP-11 dimer formation as a model for heme-protein protein interactions showed that dimer formation is favored toward more neutral pH and favored when assisted by salt bridges ( e.g. , NH 4 + , Na + and K + vs. H + ). Inspection of the dimer mobility profiles (2+ and 3+ charge states) showed a high degree of structural heterogeneity as a function of the solution pH and ion form; the observation of common mobility bands suggest that the different salt bridges can stabilize similar structural motifs. In addition, the salt bridge influence on the MP-11 dimer formations was measured using collision induced dissociation and showed a strong dependence with the type of salt bridge ( i.e. , a CE 50 of 10.0, 11.5, 11.8 and 13.0 eV was observed for [2M + H] 3+ , [2M − H + NH 4 ] 3+ , [2M + Na] 3+ and [2M + K] 3+ , respectively). Measurements of the dimer equilibrium constant showed that the salt bridge interactions increase the binding strength of the dimeric species. 

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2. Peptide Sequence Influence on the Conformational Dynamics and DNA binding of the Intrinsically Disordered AT-Hook 3 Peptide

   https://doi.org/10.1038/s41598-018-28956-z  

   Garabedian, Alyssa ; Bolufer, Alexander ; Leng, Fenfei ; Fernandez-Lima, Francisco ( July 2018 , Scientific Reports)

   The intrinsically disordered ATHP3 was studied at native conditions and in complex with DNA using single amino acid substitutions and high-resolution ion mobility spectrometry coupled to mass spectrometry (trapped IMS-MS). Results showed that ATHP3 can exist in multiple conformations at native conditions (at least 10 conformers were separated), with a variety of prolinecis/transorientations, side chain orientations and protonation sites. When in complex with AT rich DNA hairpins, the -RGRP- core is essential for stabilizing the ATHP3: DNA complex. In particular, the arginine in the sixth position plays an important role during binding to AT-rich regions of hairpin DNA, in good agreement with previous NMR and X-ray data. Mobility based correlation matrices are proposed as a way to reveal differences in structural motifs across the peptide mutants based on the conformational space and relative conformer abundance.

    

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3. Zinc Alters the Supramolecular Organization of Nucleic Acid Complexes with Full-Length TIA1

   Yizhuo Yang, Keith J. ( January 2023 , bioRxiv)

   na (Ed.)

   T-Cell Intracellular Antigen-1 (TIA1) is a 43 kDa multi-domain RNA-binding protein involved in stress granule formation during eukaryotic stress response, and has been implicated in neurodegenerative diseases including Welander distal myopathy and amyotrophic lateral sclerosis. TIA1 contains three RNA recognition motifs (RRMs), which are capable of binding nucleic acids and a C-terminal Q/N-rich prion-related domain (PRD) which has been variously described as intrinsically disordered or prion inducing and is believed to play a role in promoting liquid-liquid phase separation connected with the assembly of stress granule formation. Motivated by the fact that our prior work shows RRMs 2 and 3 are well-ordered in an oligomeric full-length form, while RRM1 and the PRD appear to phase separate, the present work addresses whether the oligomeric form is functional and competent for binding, and probes the consequences of nucleic acid binding for oligomerization and protein conformation change. New SSNMR data show that ssDNA binds to full-length oligomeric TIA1 primarily at the RRM2 domain, but also weakly at the RRM3 domain, and Zn2+ binds primarily to RRM3. Binding of Zn2+ and DNA was reversible for the full-length wild type oligomeric form, and did not lead to formation of amyloid fibrils, despite the presence of the C-terminal prion-related domain. While TIA1:DNA complexes appear as long “daisy chained” structures, the addition of Zn2+ caused the structures to collapse. We surmise that this points to a regulatory role for Zn2+. By occupying various “half” binding sites on RRM3 Zn2+ may shift the nucleic acid binding off RRM3 and onto RRM2. More importantly, the use of different half sites on different monomers may introduce a mesh of crosslinks in the supramolecular complex rendering it compact and markedly reducing the access to the nucleic acids (including transcripts) from solution. 

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4. Comparison of the pH‐dependent formation of His and Cys heptapeptide complexes of nickel(II), copper(II), and zinc(II) as determined by ion mobility‐mass spectrometry

   https://doi.org/10.1002/jms.4489  

   Yousef, Enas N. ; Angel, Laurence A. ( February 2020 , Journal of Mass Spectrometry)

   The analog methanobactin (amb) peptide with the sequence ac‐His₁‐Cys₂‐Gly₃‐Pro₄‐Tyr₅‐His₆‐Cys₇(amb_5A) will bind the metal ions of zinc, nickel, and copper. To further understand how amb_5Abinds these metals, we have undertaken a series of studies of structurally related heptapeptides where one or two of the potential His or Cys binding sites have been replaced by Gly, or the C‐terminus has been blocked by amidation. The studies were designed to compare how these metals bind to these sequences in different pH solutions of pH 4.2 to 10 and utilized native electrospray ionization (ESI) with ion mobility‐mass spectrometry (IM‐MS) which allows for the quantitative analysis of the charged species produced during the reactions. The native ESI conditions were chosen to conserve as much of the solution‐phase behavior of the amb peptides as possible and an analysis of how the IM‐MS results compare with the expected solution‐phase behavior is discussed. The oligopeptides studied here have applications for tag‐based protein purification methods, as therapeutics for diseases caused by elevated metal ion levels or as inhibitors for metal‐protein enzymes such as matrix metalloproteinases.

    

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5. Screening of Minimalist Noncanonical Sites in Duplex DNA and RNA Reveals Context and Motif‐Selective Binding by Fluorogenic Base Probes

   https://doi.org/10.1002/chem.202103616  

   Liang, Yufeng ; Miao, Shiqin ; Mao, Jie ; Devari, Shekaraiah ; Gonzalez, Maricarmen ; Bong, Dennis ( November 2021 , Chemistry – A European Journal)

   We hypothesize that programmable hybridization to noncanonical nucleic acid motifs may be achieved by macromolecular display of binders to individual noncanonical pairs (NCPs). As each recognition element may individually have weak binding to an NCP, we developed a semi‐rational approach to detect low affinity interactions between selected nitrogenous bases and noncanonical sites in duplex DNA and RNA. A set of fluorogenic probes was synthesized by coupling abiotic (triazines, pyrimidines) and native RNA bases to thiazole orange (TO) dye. This probe library was screened against duplex nucleic acid substrates bearing single abasic, single NCP, and tandem NCP sites. Probe engagement with NCP sites was reported by 100–1000× fluorescence enhancement over background. Binding is strongly context‐dependent, reflective of both molecular recognition and stability: less stable motifs are more likely to bind a synthetic probe. Further, DNA and RNA substrates exhibit entirely different abasic and single NCP binding profiles. While probe binding in the abasic and single NCP screens was monotonous, much richer binding profiles were observed with the screen of tandem NCP sites in RNA, in part due to increased steric accessibility. In addition to known binding interactions between the triazine melamine (M) and T/U sites, the NCP screens identified new targeting elements for pyrimidine‐rich motifs in single NCPs and 2×2 internal bulges. We anticipate that semi‐rational approaches of this type will lead to programmable noncanonical hybridization strategies at the macromolecular level.

    

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