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1. Secondary amine selective Petasis (SASP) bioconjugation

   https://doi.org/10.1039/C9SC04697F  

   Sim, Yonnette E.; Nwajiobi, Ogonna; Mahesh, Sriram; Cohen, Ryan D.; Reibarkh, Mikhail Y.; Raj, Monika (January 2020, Chemical Science)

   Selective modification of proteins enables synthesis of antibody-drug conjugates, cellular drug delivery and construction of new materials. Many groups have developed methods for selective N-terminal modification without affecting the side chain of lysine by judicious pH control. This is due to lower basicity of the N-terminus relative to lysine side chains. But none of the methods are capable of selective modification of secondary amines or N-terminal proline, which has similar basicity as lysine. Here, we report a secondary amine selective Petasis (SASP) reaction for selective bioconjugation at N-terminal proline. We exploited the ability of secondary amines to form highly electrophilic iminium ions with aldehydes, which rapidly reacted with nucleophilic organoboronates, resulting in robust labeling of N-terminal proline under biocompatible conditions. This is the first time the Petasis reaction has been utilized for selective modification of secondary amines on completely unprotected peptides and proteins under physiological conditions. Peptide screening results showed that the reaction is highly selective for N-terminal proline. There are no other chemical methods reported in literature that are selective for N-terminal proline in both peptides and proteins. This is a multicomponent reaction leading to the synthesis of doubly functionalized bioconjugates in one step that can be difficult to achieve using other methods. The key advantage of the SASP reaction includes its high chemoselective and stereoselective (>99% de) nature, and it affords dual labeled proteins in one pot. The broad utility of this bioconjugation is highlighted for a variety of peptides and proteins, including aldolase and creatine kinase. 

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2. N-Terminal Proline Editing for the Synthesis of Peptides with Mercaptoproline and Selenoproline: Mechanistic Insights Lead to Greater Efficiency in Proline Native Chemical Ligation

   https://doi.org/10.1021/acschembio.3c00705  

   Ludwig, Brice A; Forbes, Christina R; Zondlo, Neal J (February 2024, ACS Chemical Biology)

   Native chemical ligation (NCL) at proline has been limited by cost and synthetic access. In addition, prior examples of NCL using mercaptoproline have exhibited stalling of the reaction after thioester exchange, due to inefficient SN acyl transfer. Herein, we develop methods, using inexpensive Boc-4R-hydroxyproline, for the solid-phase synthesis of peptides containing N-terminal 4R-mercaptoproline and 4R-selenoproline. The synthesis proceeds via proline editing on the N-terminus of fully synthesized peptides on the solid phase, converting an N-terminal Boc-4R-hydroxyproline to the 4S-bromoproline, followed by SN2 reaction with potassium thioacetate or selenobenzoic acid. After cleavage from the resin and deprotection, peptides with functionalized N-terminal proline amino acids were obtained. NCL reactions with mercaptoproline proceeded slowly under standard NCL conditions, with the S-acyl transthioesterification intermediate observed as a major species. Computational investigations indicated that the bicyclic intermediates and transition states for SN acyl transfer are sufficiently low in energy (10-15 kcal mol–1 above starting material) that ring strain cannot explain slow SN acyl transfer. Instead, the bicyclic zwitterionic tetrahedral intermediate has a low barrier for reversion to the S-acyl intermediate, causing reversion to the thioester (reverse reaction) to occur preferentially over elimination to generate the amide (forward reaction). We hypothesized that a buffer capable of general acid and/or general base catalysis could promote SN acyl transfer, and thus achieve greater efficiency in proline NCL. In the presence of 2 M imidazole at pH 6.8, NCL with mercaptoproline proceeded efficiently to generate the peptide with a native amide bond. NCL with selenoproline also proceeded efficiently to generate the desired products when a thiophenol thioester was employed as a ligation partner. After desulfurization or deselenization, the products obtained were identical to those synthesized directly, confirming that the solid-phase proline editing reactions proceeded stereospecifically and without epimerization. 

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3. Effects of terminal tripeptide units on mechanical properties of collagen triple helices

   https://doi.org/10.1016/j.eml.2023.102075  

   Masrouri, Milad; Qin, Zhao (November 2023, Extreme Mechanics Letters)

   Collagen, a vital protein that provides strength to various body tissues, has a triple helix structure containing three polypeptide chains. The chains are composed mostly of a tripeptide of glycine (G), proline (P), and hydroxyproline (O). Using molecular dynamics simulations and theoretical analysis, the study examines the mechanical response of collagen triple helix structures, made up of three different tripeptide units, when subjected to different fracture loading modes. The results show that collagen with GPO tripeptide units at their C-terminal are mechanically stronger than the POG and OGP units with a single amino-acid frame shift. Our work shows that the N-terminal has less effect on collagen fracture than the C-terminal. The differences in mechanical response are explained by the heterogenous rigidity of the amino acid backbone and the resulting shear lag effect near the terminal. The findings have potential applications in developing tough synthetic collagen for building materials and may stimulate further studies on the connection between terminal repeats and the mechanical-thermal behavior of other structural proteins such as silk, elastin, fibrin, and keratin. 

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4. Helical twists and β‐turns in structures at serine–proline sequences: Stabilization of cis ‐proline and type VI β‐turns via C–H/O interactions

   https://doi.org/10.1002/prot.26701  

   Oven, Harrison_C; Yap, Glenn_P_A; Zondlo, Neal_J (May 2024, Proteins: Structure, Function, and Bioinformatics)

   Abstract Structures at serine‐proline sites in proteins were analyzed using a combination of peptide synthesis with structural methods and bioinformatics analysis of the PDB. Dipeptides were synthesized with the proline derivative (2S,4S)‐(4‐iodophenyl)hydroxyproline [hyp(4‐I‐Ph)]. The crystal structure of Boc‐Ser‐hyp(4‐I‐Ph)‐OMe had two molecules in the unit cell. One molecule exhibitedcis‐proline and a type VIa2 β‐turn (BcisD). Thecis‐proline conformation was stabilized by a C–H/O interaction between Pro C–H_αand the Ser side‐chain oxygen. NMR data were consistent with stabilization ofcis‐proline by a C–H/O interaction in solution. The other crystallographically observed molecule hadtrans‐Pro and both residues in the PPII conformation. Two conformations were observed in the crystal structure of Ac‐Ser‐hyp(4‐I‐Ph)‐OMe, with Ser adopting PPII in one and the β conformation in the other, each with Pro in the δ conformation andtrans‐Pro. Structures at Ser‐Pro sequences were further examined via bioinformatics analysis of the PDB and via DFT calculations. Ser‐Pro versus Ala–Pro sequences were compared to identify bases for Ser stabilization of local structures. C–H/O interactions between the Ser side‐chain O_γand Pro C–H_αwere observed in 45% of structures with Ser‐cis‐Pro in the PDB, with nearly all Ser‐cis‐Pro structures adopting a type VI β‐turn. 53% of Ser‐trans‐Pro sequences exhibited main‐chain CO_i•••HN_i+3or CO_i•••HN_i+4hydrogen bonds, with Ser as theiresidue and Pro as thei + 1 residue. These structures were overwhelmingly either type I β‐turns or N‐terminal capping motifs on α‐helices or 3₁₀‐helices. These results indicate that Ser‐Pro sequences are particularly potent in favoring these structures. In each, Ser is in either the PPII or β conformation, with the Ser O_γcapable of engaging in a hydrogen bond with the amide N–H of thei + 2 (type I β‐turn or 3₁₀‐helix; Serχ₁t) ori + 3 (α‐helix; Serχ₁g⁺) residue. Non‐prolinecisamide bonds can also be stabilized by C–H/O interactions. 

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5. GROWTH POLE RING protein forms a 200-nm-diameter ring structure essential for polar growth and rod shape in Agrobacterium tumefaciens

   https://doi.org/10.1073/pnas.1905900116  

   Zupan, J. R.; Grangeon, R.; Robalino-Espinosa, J. S.; Garnica, N.; Zambryski, P. (May 2019, Proceedings of the National Academy of Sciences)

   Polar growth in Agrobacterium pirates and repurposes well-known bacterial cell cycle proteins, such as FtsZ, FtsA, PopZ, and PodJ. Here we identify a heretofore unknown protein that we name GROWTH POLE RING (GPR) due to its striking localization as a hexameric ring at the growth pole during polar growth. GPR also localizes at the midcell late in the cell cycle just before division, where it is then poised to be precisely localized at new growth poles in sibling cells. GPR is 2,115 aa long, with two N-terminal transmembrane domains placing the bulk of the protein in the cytoplasm, N- and C-terminal proline-rich disordered regions, and a large 1,700-aa central region of continuous α-helical domains. This latter region contains 12 predicted adjacent or overlapping apolipoprotein domains that may function to sequester lipids during polar growth. Stable genetic deletion or riboswitch-controlled depletion results in spherical cells that grow poorly; thus, GPR is essential for wild-type growth and morphology. As GPR has no predicted enzymatic domains and it forms a distinct 200-nm-diameter ring, we propose that GPR is a structural component of an organizing center for peptidoglycan and membrane syntheses critical for cell envelope formation during polar growth. GPR homologs are found in numerous Rhizobiales; thus, our results and proposed model are fundamental to understanding polar growth strategy in a variety of bacterial species. 

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