Low-cost synthesis of peptide libraries and their use for binding studies via temperature-related intensity change.

Protein-peptide interactions are involved in many fundamental cellular functions and constitute promising drug targets. Here, we provide a detailed protocol for the cost-effective preparation of a cellulose-based solid support for synthesis of nanoscale to micromolar-scale peptide libraries. Their subsequent use for high-throughput protein interaction screening as well as affinity determination in solution provides binding data for thousands of unique peptides with a turnover of 1 to 2 weeks, thereby facilitating assessment and development of high-affinity binders.

High-throughput determination of protein affinities using unmodified peptide libraries in nanomolar scale.

Protein-protein interactions (PPIs) are of fundamental importance for our understanding of physiology and pathology. PPIs involving short, linear motifs play a major role in immunological recognition, signaling, and regulation and provide attractive starting points for pharmaceutical intervention. Yet, state-of-the-art protein-peptide affinity determination approaches exhibit limited throughput and sensitivity, often resulting from ligand immobilization, labeling, or synthesis.

RanGTPase regulates the interaction between the inner nuclear membrane proteins, Samp1 and Emerin.

Samp1, spindle associated membrane protein 1, is a type II integral membrane protein localized in the inner nuclear membrane. Recent studies have shown that the inner nuclear membrane protein, Emerin and the small monomeric GTPase, Ran are direct binding partners of Samp1. Here we addressed the question whether Ran could regulate the interaction between Samp1 and Emerin in the inner nuclear membrane.

Chaperonin-Assisted Protein Folding: Relative Population of Asymmetric and Symmetric GroEL:GroES Complexes.

The chaperonin GroEL, a cylindrical complex consisting of two stacked heptameric rings, and its lid-like cofactor GroES form a nano-cage in which a single polypeptide chain is transiently enclosed and allowed to fold unimpaired by aggregation. GroEL and GroES undergo an ATP-regulated interaction cycle that serves to close and open the folding cage. Recent reports suggest that the presence of non-native substrate protein alters the GroEL/ES reaction by shifting it from asymmetric to symmetric complexes.

Active cage mechanism of chaperonin-assisted protein folding demonstrated at single-molecule level.

The cylindrical chaperonin GroEL and its lid-shaped cofactor GroES of Escherichia coli have an essential role in assisting protein folding by transiently encapsulating non-native substrate in an ATP-regulated mechanism. It remains controversial whether the chaperonin system functions solely as an infinite dilution chamber, preventing off-pathway aggregation, or actively enhances folding kinetics by modulating the folding energy landscape. Here we developed single-molecule approaches to distinguish between passive and active chaperonin mechanisms.

GroEL/ES chaperonin modulates the mechanism and accelerates the rate of TIM-barrel domain folding.

The GroEL/ES chaperonin system functions as a protein folding cage. Many obligate substrates of GroEL share the (βα)8 TIM-barrel fold, but how the chaperonin promotes folding of these proteins is not known. Here, we analyzed the folding of DapA at peptide resolution using hydrogen/deuterium exchange and mass spectrometry.