Multilayered allosteric modulation of coupled folding and binding by phosphorylation, peptidyl-prolyl cis/trans isomerization, and diversity of interaction partners.

Intrinsically disordered proteins (IDPs) play key roles in cellular regulation, including signal transduction, transcription, and cell-cycle control. Accordingly, IDPs can commonly interact with numerous different target proteins, and their interaction networks are expected to be highly regulated. However, many of the underlying regulatory mechanisms have remained unclear.

Labeling of Proteins for Single-Molecule Fluorescence Spectroscopy.

Single-molecule fluorescence spectroscopy has become an important technique for studying the conformational dynamics and folding of proteins. A key step for performing such experiments is the availability of high-quality samples. This chapter describes a simple and widely applicable strategy for preparing proteins that are site-specifically labeled with a donor and an acceptor dye for single-molecule Förster resonance energy transfer (FRET) experiments.

The effect of fatty diacid acylation of human PYY on Y receptor potency and half-life in minipigs.

Peptides are notoriously known to display very short in vivo half-lives often measured in minutes which in many cases greatly reduces or eliminates sufficient in vivo efficacy. To obtain long half-lives allowing for up to once-weekly dosing regimen, fatty acid acylation (lipidation) have been used to non-covalently associate the peptide to serum albumin thus serving as a circulating depot. This approach is generally considered in the scientific and patent community as a standard approach to protract almost any given peptide.

SARS-CoV-2 Neutralizing Antibody Responses towards Full-Length Spike Protein and the Receptor-Binding Domain.

Tools to monitor SARS-CoV-2 transmission and immune responses are needed. We present a neutralization ELISA to determine the levels of Ab-mediated virus neutralization and a preclinical model of focused immunization strategy. The ELISA is strongly correlated with the elaborate plaque reduction neutralization test (ρ = 0.

Polyelectrolyte interactions enable rapid association and dissociation in high-affinity disordered protein complexes.

Highly charged intrinsically disordered proteins can form complexes with very high affinity in which both binding partners fully retain their disorder and dynamics, exemplified by the positively charged linker histone H1.0 and its chaperone, the negatively charged prothymosin α. Their interaction exhibits another surprising feature: The association/dissociation kinetics switch from slow two-state-like exchange at low protein concentrations to fast exchange at higher, physiologically relevant concentrations.

Depletion interactions modulate the binding between disordered proteins in crowded environments.

Intrinsically disordered proteins (IDPs) abound in cellular regulation. Their interactions are often transitory and highly sensitive to salt concentration and posttranslational modifications. However, little is known about the effect of macromolecular crowding on the interactions of IDPs with their cellular targets.

Structure-Guided Design of a Peptide Lock for Modular Peptide Binders.

Peptides play an important role in intermolecular interactions and are frequent analytes in diagnostic assays, also as unstructured, linear epitopes in whole proteins. Yet, due to the many different sequence possibilities even for short peptides, classical selection of binding proteins from a library, one at a time, is not scalable to proteomes. However, moving away from selection to a rational assembly of preselected modules binding to predefined linear epitopes would split the problem into smaller parts.

Distinct G protein-coupled receptor phosphorylation motifs modulate arrestin affinity and activation and global conformation.

Cellular functions of arrestins are determined in part by the pattern of phosphorylation on the G protein-coupled receptors (GPCRs) to which arrestins bind. Despite high-resolution structural data of arrestins bound to phosphorylated receptor C-termini, the functional role of each phosphorylation site remains obscure. Here, we employ a library of synthetic phosphopeptide analogues of the GPCR rhodopsin C-terminus and determine the ability of these peptides to bind and activate arrestins using a variety of biochemical and biophysical methods.

Transition path times of coupled folding and binding reveal the formation of an encounter complex.

The association of biomolecules is the elementary event of communication in biology. Most mechanistic information of how the interactions between binding partners form or break is, however, hidden in the transition paths, the very short parts of the molecular trajectories from the encounter of the two molecules to the formation of a stable complex. Here we use single-molecule spectroscopy to measure the transition path times for the association of two intrinsically disordered proteins that form a folded dimer upon binding.

Charge Interactions Can Dominate Coupled Folding and Binding on the Ribosome.

Interactions between emerging nascent polypeptide chains and the ribosome can modulate cotranslational protein folding. However, it has remained unclear how such interactions can affect the binding of nascent chains to their cellular targets. We thus investigated on the ribosome the interaction between two intrinsically disordered proteins of opposite charge, ACTR and NCBD, which form a high-affinity complex in a coupled folding-and-binding reaction.

A proline switch explains kinetic heterogeneity in a coupled folding and binding reaction.

The interactions of intrinsically disordered proteins (IDPs) with their molecular targets are essential for the regulation of many cellular processes. IDPs can perform their functions while disordered, and they may fold to structured conformations on binding. Here we show that the cis/trans isomerization of peptidyl-prolyl bonds can have a pronounced effect on the interactions of IDPs.

Internal friction in an intrinsically disordered protein-Comparing Rouse-like models with experiments.

Internal friction is frequently found in protein dynamics. Its molecular origin however is difficult to conceptualize. Even unfolded and intrinsically disordered polypeptide chains exhibit signs of internal friction despite their enormous solvent accessibility.

The Three-Fold Axis of the HIV-1 Capsid Lattice Is the Species-Specific Binding Interface for TRIM5α.

Rhesus TRIM5α (rhTRIM5α) potently restricts replication of human immunodeficiency virus type 1 (HIV-1). Restriction is mediated through direct binding of the C-terminal B30.2 domain of TRIM5α to the assembled HIV-1 capsid core.

Combining short- and long-range fluorescence reporters with simulations to explore the intramolecular dynamics of an intrinsically disordered protein.

Intrinsically disordered proteins (IDPs) are increasingly recognized as a class of molecules that can exert essential biological functions even in the absence of a well-defined three-dimensional structure. Understanding the conformational distributions and dynamics of these highly flexible proteins is thus essential for explaining the molecular mechanisms underlying their function. Single-molecule fluorescence spectroscopy in combination with Förster resonance energy transfer (FRET) is a powerful tool for probing intramolecular distances and the rapid long-range distance dynamics in IDPs.

Rapid Microfluidic Dilution for Single-Molecule Spectroscopy of Low-Affinity Biomolecular Complexes.

To enable the investigation of low-affinity biomolecular complexes with confocal single-molecule spectroscopy, we have developed a microfluidic device that allows a concentrated sample to be diluted by up to five orders of magnitude within milliseconds, at the physical limit dictated by diffusion. We demonstrate the capabilities of the device by studying the dissociation kinetics and structural properties of low-affinity protein complexes using single-molecule two-color and three-color Förster resonance energy transfer (FRET). We show that the versatility of the device makes it suitable for studying complexes with dissociation constants from low nanomolar up to 10 μm, thus covering a wide range of biomolecular interactions.

Rapid Microfluidic Double-Jump Mixing Device for Single-Molecule Spectroscopy.

We introduce a microfluidic double-jump mixing device for investigating rapid biomolecular kinetics with confocal single-molecule spectroscopy. This device enables nonequilibrium dynamics to be probed, e.g.

Single-molecule electrometry.

Mass and electrical charge are fundamental properties of biological macromolecules. Although molecular mass has long been determined with atomic precision, a direct and precise determination of molecular charge remains an outstanding challenge. Here we report high-precision (<1e) measurements of the electrical charge of molecules such as nucleic acids, and globular and disordered proteins in solution.

Generation of Fluorogen-Activating Designed Ankyrin Repeat Proteins (FADAs) as Versatile Sensor Tools.

Fluorescent probes constitute a valuable toolbox to address a variety of biological questions and they have become irreplaceable for imaging methods. Commonly, such probes consist of fluorescent proteins or small organic fluorophores coupled to biological molecules of interest. Recently, a novel class of fluorescence-based probes, fluorogen-activating proteins (FAPs), has been reported.

Gas-Phase FRET Efficiency Measurements To Probe the Conformation of Mass-Selected Proteins.

Electrospray ionization and mass spectrometry have revolutionized the chemical analysis of biological molecules, including proteins. However, the correspondence between a protein's native structure and its structure in the mass spectrometer (where it is gaseous) remains unclear. Here, we show that fluorescence (Förster) resonance energy transfer (FRET) measurements combined with mass spectrometry provides intramolecular distance constraints in gaseous, ionized proteins.

Visualization of HRas Domains in the Plasma Membrane of Fibroblasts.

The plasma membrane is a highly complex, organized structure where the lateral organization of signaling proteins is tightly regulated. In the case of Ras proteins, it has been suggested that the differential activity of the various isoforms is due to protein localization in separate membrane compartments. To date, direct visualization of such compartmentalization has been achieved only by electron microscopy on membrane sheets.

Single-molecule spectroscopy reveals polymer effects of disordered proteins in crowded environments.

Intrinsically disordered proteins (IDPs) are involved in a wide range of regulatory processes in the cell. Owing to their flexibility, their conformations are expected to be particularly sensitive to the crowded cellular environment. Here we use single-molecule Förster resonance energy transfer to quantify the effect of crowding as mimicked by commonly used biocompatible polymers.

Intramolecular distances and dynamics from the combined photon statistics of single-molecule FRET and photoinduced electron transfer.

Single-molecule Förster resonance energy transfer (FRET) and photoinduced electron transfer (PET) have developed into versatile and complementary methods for probing distances and dynamics in biomolecules. Here we show that the two methods can be combined in one molecule to obtain both accurate distance information and the kinetics of intramolecular contact formation. In a first step, we show that the fluorescent dyes Alexa 488 and Alexa 594, which are frequently used as a donor and acceptor for single-molecule FRET, are also suitable as PET probes with tryptophan as a fluorescence quencher.

Ligand binding to heme proteins: a comparison of cytochrome c variants with globins.

We have studied the binding of carbon monoxide (CO) in mutants of Cyt c having its methionine at position 80 replaced by alanine, aspartate, and arginine, so that the sixth coordination is available for ligand binding. We have employed Fourier transform infrared (FTIR) photolysis difference spectroscopy to examine interactions of the heme-bound and photolyzed CO (and also nitric oxide, NO) in the small heme pocket created by the mutations. By using FTIR temperature derivative spectroscopy (TDS) and nanosecond flash photolysis, the enthalpy barrier distributions for CO rebinding were determined.