Multi phosphorylated peptides are key tools in understanding the biological roles of protein phosphorylation patterns. In this work, we focused on multi phosphorylated peptides with over four, clustered, phosphorylation sites that are termed herein heavily phosphorylated peptides (HPPs). The synthesis of heavily phosphorylated peptides is extremely difficult and requires the use of a wide temperature range. Standard peptide synthesizers are incapable of both cooling and heating, which impedes the automated synthesis of those peptides. Herein, we used the oligosaccharide synthesizer Glyconeer 2.1 to develop a protocol for the automated synthesis of heavily phosphorylated peptides. The Glyconeer 2.1 is able to both cool and heat, which enabled the development of highly controlled coupling and deprotection conditions that were used for the automated synthesis of four different heavily phosphorylated peptides with five or more, clustered, phosphorylation sites. Our approach paves the way for an easy automated synthesis of a variety of heavily phosphorylated peptides.
Intrinsically disordered regions (IDRs) in proteins are highly abundant, but they are still commonly viewed as long stretches of polar, solvent-accessible residues. Here we show that the disordered C-terminal domain (CTD) of HIV-1 Rev has two subregions that carry out two distinct complementary roles of regulating protein oligomerization and contributing to stability. We propose that this takes place through a delicate balance between charged and hydrophobic residues within the IDR. This means that mutations in this region, as well as the known mutations in the structured region of the protein, can affect protein function. We suggest that IDRs in proteins should be divided into subdomains similarly to structured regions, rather than being viewed as long flexible stretches.
Preparing phosphorylated peptides with multiple adjacent
phosphorylations
is synthetically difficult, leads to β-elimination, results
in low yields, and is extremely slow. We combined synthetic chemical
methodologies with computational studies and engineering approaches
to develop a strategy that takes advantage of fast stirring, high
temperature, and a very low concentration of 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU) to produce multiphosphorylated peptides at an extremely rapid
time and high purity.
Photocleavage from
polystyrene beads is a pivotal reaction for solid phase synthesis that relies on
photolabile linkers. The photocleavage, usually performed by batch irradiation,
suffers from incomplete and slow cleavage. To overcome these issues, continuous
flow and high-energy lamps are frequently used, but these setups are hazardous,
technically challenging, and expensive. We developed a photocleavage approach
that relies on a benchtop LED lamp and magnetic stirring. In this approach, we
crush the beads instead of keeping their integrity to increase the yield of
photocleavage. This approach proved very efficient for photocleavage of
protected oligosaccharides.
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