23 research outputs found
Calculation of the relative metastabilities of proteins in subcellular compartments of Saccharomyces cerevisiae
[abridged] Background: The distribution of chemical species in an open system
at metastable equilibrium can be expressed as a function of environmental
variables which can include temperature, oxidation-reduction potential and
others. Calculations of metastable equilibrium for various model systems were
used to characterize chemical transformations among proteins and groups of
proteins found in different compartments of yeast cells.
Results: With increasing oxygen fugacity, the relative metastability fields
of model proteins for major subcellular compartments go as mitochondrion,
endoplasmic reticulum, cytoplasm, nucleus. In a metastable equilibrium setting
at relatively high oxygen fugacity, proteins making up actin are predominant,
but those constituting the microtubule occur with a low chemical activity. A
reaction sequence involving the microtubule and spindle pole proteins was
predicted by combining the known intercompartmental interactions with a
hypothetical program of oxygen fugacity changes in the local environment. In
further calculations, the most-abundant proteins within compartments generally
occur in relative abundances that only weakly correspond to a metastable
equilibrium distribution. However, physiological populations of proteins that
form complexes often show an overall positive or negative correlation with the
relative abundances of proteins in metastable assemblages.
Conclusions: This study explored the outlines of a thermodynamic description
of chemical transformations among interacting proteins in yeast cells. The
results suggest that these methods can be used to measure the degree of
departure of a natural biochemical process or population from a local minimum
in Gibbs energy.Comment: 32 pages, 7 figures; supporting information is available at
http://www.chnosz.net/yeas
Cyclizing disulfide-rich peptides using sortase A
Sortase A (SrtA) is an enzyme obtained from Staphylococcus aureus that catalyzes site-specific transpeptidation of surface proteins to the bacterial cell membrane. SrtA recognizes an LPXTG amino acid motif and cleaves between the Thr and Gly to form a thioester-linked acyl–enzyme intermediate. The intermediate is resolved in the presence of a nucleophilic N-terminal polyglycine resulting in ligation of the acyl donor to the polyglycine acceptor. Here we describe the application of SrtA as a tool for the cyclization of disulfide-rich peptides. Reactions are typically tailored to each disulfide-rich peptide with optimal conditions producing yields of 40–50% cyclized peptide
Prevention of aspartimide formation during peptide synthesis using cyanosulfurylides as carboxylic acid-protecting groups
A lanthipeptide library used to identify a protein-protein interaction inhibitor
We describe the production and screening of a genetically encoded library of 106 lanthipeptides in Escherichia coli using the substrate tolerant lanthipeptide synthetase ProcM. This plasmid-encoded library was combined with a bacterial reverse two-hybrid system for the interaction of the HIV p6 protein with the UEV domain of the human TSG101 protein, a critical protein-protein interaction for HIV budding from infected cells. Using this approach, we identified an inhibitor of this interaction from the lanthipeptide library, whose activity was verified in vitro and in cell-based virus-like particle budding assays. Given the variety of lanthipeptide backbone scaffolds that may be produced with ProcM, this method may be used for the generation of genetically encoded libraries of natural product-like lanthipeptides containing significant structural diversity. Such libraries may be combined with any cell-based assay for the identification of lanthipeptides with new biological activities
NMR relaxation analysis of pharmaceutically active peptides
Nuclear spin relaxation (NSR) is a powerful approach for studying dynamics at the ps-ns timescale, and is typically used to characterize fundamental biophysical phenomena such as bond vibrations and fluctuations, which affect the activity of the molecule in question. Here, this chapter will look to the application of NSR to study peptides, which are short chains of amino acids and have shown promise as modalities in drug design. This chapter will begin with a brief description of theoretical and practical aspects related to the use of NSR, such as experimental considerations during data acquisition and processing. As an example of this approach for studying peptide dynamics, this chapter will step through a case study that examines the effect of backbone cyclization on the dynamics of polycyclic disulfide-rich peptides. This case study will focus on a cyclic and linear variant of a promising drug scaffold isolated from sunflower seeds called SFTI-1 (sunflower trypsin inhibitor-1), which is a naturally backbone-cyclic peptide that comprises one cross-bracing disulfide bond
Dual-targeting anti-angiogenic cyclic peptides as potential drug leads for cancer therapy
Molecular cloning and sequence characterization of common carp (Cyprinus carpio) integrin β1 (ITGβ1) and its temporal expression in response to CyHV-3
Exo-endocytic trafficking and the septin-based diffusion barrier are required for the maintenance of Cdc42p polarization during budding yeast asymmetric growth
The small GTPase Cdc42p is a master regulator of cell polarity. We analyzed Cdc42p localization using yeast mutants and found that endo-exocytic trafficking and septin-based diffusion barrier synergistically control Cdc42p polarization during asymmetric cell growth