De Novo Sequencing of Synthetic Bis-cysteine Peptide Macrocycles Enabled by "Chemical Linearization" of Compound Mixtures.

A "chemical linearization" approach was applied to synthetic peptide macrocycles to enable their de novo sequencing from mixtures using nanoliquid chromatography-tandem mass spectrometry (nLC-MS/MS). This approach─previously applied to individual macrocycles but not to mixtures─involves cleavage of the peptide backbone at a defined position to give a product capable of generating sequence-determining fragment ions. Here, we first established the compatibility of "chemical linearization" by Edman degradation with a prominent macrocycle scaffold based on bis-Cys peptides cross-linked with the m-xylene linker, which are of major significance in therapeutics discovery. Then, using macrocycle libraries of known sequence composition, the ability to recover accurate de novo assignments to linearized products was critically tested using performance metrics unique to mixtures. Significantly, we show that linearized macrocycles can be sequenced with lower recall compared to linear peptides but with similar accuracy, which establishes the potential of using "chemical linearization" with synthetic libraries and selection procedures that yield compound mixtures. Sodiated precursor ions were identified as a significant source of high-scoring but inaccurate assignments, with potential implications for improving automated de novo sequencing more generally.

Directed co-evolution of interacting protein-peptide pairs by compartmentalized two-hybrid replication (C2HR).

Directed evolution methodologies benefit from read-outs quantitatively linking genotype to phenotype. We therefore devised a method that couples protein-peptide interactions to the dynamic read-out provided by an engineered DNA polymerase. Fusion of a processivity clamp protein to a thermostable nucleic acid polymerase enables polymerase activity and DNA amplification in otherwise prohibitive high-salt buffers. Here, we recapitulate this phenotype by indirectly coupling the Sso7d processivity clamp to Taq DNA polymerase via respective fusion to a high affinity and thermostable interacting protein-peptide pair. Escherichia coli cells co-expressing protein-peptide pairs can directly be used in polymerase chain reactions to determine relative interaction strengths by the measurement of amplicon yields. Conditional polymerase activity is further used to link genotype to phenotype of interacting protein-peptide pairs co-expressed in E. coli using the compartmentalized self-replication directed evolution platform. We validate this approach, termed compartmentalized two-hybrid replication, by selecting for high-affinity peptides that bind two model protein partners: SpyCatcher and the large fragment of NanoLuc luciferase. We further demonstrate directed co-evolution by randomizing both protein and peptide components of the SpyCatcher-SpyTag pair and co-selecting for functionally interacting variants.

Methods and Approaches for the Solid-Phase Synthesis of Peptide Alcohols.

Many methods have been developed for attaching an alcohol functionality to a solid support. However, not all of these methods are used to obtain peptide alcohols. In this Minireview, we will discuss several of the most important methods and approaches for the synthesis of peptide alcohols and the attachment of hydroxy groups to a solid support for the synthesis of cyclic peptides. Some of the methods include the use of functionalized Wang resin and the attachment of an alcohol to an enol ether resin. We also discuss the use of the chlorotrityl resin, one of the most common linkers used to obtain peptide alcohols. In addition, we outline the recently developed resins with the Rink, Ramage and Sieber handles. The majority of these methods have been used to synthesize many important drugs, such as octreotide and the antibiotic peptaibols.

Functionalized Resins for the Synthesis of Peptide Alcohols.

Peptide alcohols are clinically important compounds that are underexplored in structure-activity relationship (SAR) studies in drug discovery. One reason for this underutilization is that current syntheses are laborious and time consuming. Herein, we describe the preparation and utility of Rink, Ramage, and Sieber-chloride resins, which enables the use of a general, easy and practical method for the attachment of fluorenylmethoxycarbonyl (Fmoc)-amino alcohols to a solid support, in the synthesis of peptide alcohols. This method is the first straightforward Fmoc/tBu synthesis of peptide alcohols starting from a pre-loaded resin. The synthesized peptide alcohols can be detached from the linkers through conventional methods. Treatment with trifluoroacetic acid (TFA) (95 %) and scavengers such as triisopropylsilane and water for 2 h is sufficient to obtain a fully deprotected peptide alcohol, while treatment with 20 % hexafluoroisopropanol in dichloromethane renders a fully protected peptide alcohol that can be further modified at the C-terminus. As examples, the new resins were used in straightforward, relatively rapid syntheses of the peptide alcohols octreotide, alamethicin, and a segment of trichogin GA IV, as well as the first synthesis of stapled peptide alcohols.

The RopGEF KARAPPO Is Essential for the Initiation of Vegetative Reproduction in Marchantia polymorpha.

Many plants can reproduce vegetatively, producing clonal progeny from vegetative cells; however, little is known about the molecular mechanisms underlying this process. Liverwort (Marchantia polymorpha), a basal land plant, propagates asexually via gemmae, which are clonal plantlets formed in gemma cups on the dorsal side of the vegetative thallus [1]. The initial stage of gemma development involves elongation and asymmetric divisions of a specific type of epidermal cell, called a gemma initial, which forms on the floor of the gemma cup [2, 3]. To investigate the regulatory mechanism underlying gemma development, we focused on two allelic mutants in which no gemma initial formed; these mutants were named karappo, meaning "empty." We used whole-genome sequencing of both mutants and molecular genetic analysis to identify the causal gene, KARAPPO (KAR), which encodes a ROP guanine nucleotide exchange factor (RopGEF) carrying a plant-specific ROP nucleotide exchanger (PRONE) catalytic domain. In vitro GEF assays showed that the full-length KAR protein and the PRONE domain have significant GEF activity toward MpROP, the only ROP GTPase in M. polymorpha. Moreover, genetic complementation experiments showed a significant role for the N- and C-terminal variable regions in gemma development. Our investigation demonstrates an essential role for KAR/RopGEF in the initiation of plantlet development from a differentiated cell, which may involve cell-polarity formation and subsequent asymmetric cell division via activation of ROP signaling, implying a similar developmental mechanism in vegetative reproduction of various land plants.