Studying DNA–protein interactions with single-molecule Förster resonance energy transfer

Single-molecule Förster resonance energy transfer (smFRET) has emerged as a powerful tool for elucidating biological structure and mechanisms on the molecular level. Here, we focus on applications of smFRET to study interactions between DNA and enzymes such as DNA and RNA polymerases. SmFRET, used as a nanoscopic ruler, allows for the detection and precise characterisation of dynamic and rarely occurring events, which are otherwise averaged out in ensemble-based experiments. In this review, we will highlight some recent developments that provide new means of studying complex biological systems either by combining smFRET with force-based techniques or by using data obtained from smFRET experiments as constrains for computer-aided modelling.     

Peptide-based covalent inhibitors of protein–protein interactions

Protein–protein interactions (PPI) are involved in all cellular processes and many represent attractive therapeutic targets. However, the frequently rather flat and large interaction areas render the identification of small molecular PPI inhibitors very challenging. As an alternative, peptide interaction motifs derived from a PPI interface can serve as starting points for the development of inhibitors. However, certain proteins remain challenging targets when applying inhibitors with a competitive mode of action. For that reason, peptide-based ligands with an irreversible binding mode have gained attention in recent years. This review summarizes examples of covalent inhibitors that employ peptidic binders and have been tested in a biological context.     

Comparative mapping of host–pathogen protein–protein interactions

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Chemoproteomic profiling of protein–metabolite interactions

Small molecule metabolites play important roles in regulating protein functions, which are acted through either covalent non-enzymatic post-translational modifications or non-covalent binding interactions. Chemical proteomic strategies can help delineate global landscapes of cellular protein–metabolite interactions and provide molecular insights about their mechanisms of action. In this review, we summarized the recent progress in developments and applications of chemoproteomic strategies to profile protein–metabolite interactions.     

Landscape of π–π and sugar–π contacts in DNA–protein interactions

There were 1765 contacts identified between DNA nucleobases or deoxyribose and cyclic (W, H, F, Y) or acyclic (R, E, D) amino acids in 672 X-ray structures of DNA–protein complexes. In this first study to compare π-interactions between the cyclic and acyclic amino acids, visual inspection was used to categorize amino acid interactions as nucleobase π–π (according to biological edge) or deoxyribose sugar–π (according to sugar edge). Overall, 54% of contacts are nucleobase π–π interactions, which involve all amino acids, but are more common for Y, F, and R, and involve all DNA nucleobases with similar frequencies. Among binding arrangements, cyclic amino acids prefer more planar (stacked) π-systems than the acyclic counterparts. Although sugar–π interactions were only previously identified with the cyclic amino acids and were found to be less common (38%) than nucleobase–cyclic amino acid contacts, sugar–π interactions are more common than nucleobase π–π contacts for the acyclic series (61% of contacts). Similar to DNA–protein π–π interactions, sugar–π contacts most frequently involve Y and R, although all amino acids adopt many binding orientations relative to deoxyribose. These DNA–protein π-interactions stabilize biological systems, by up to approximately ?40 kJ mol^?1 for neutral nucleobase or sugar–amino acid interactions, but up to approximately ?95 kJ mol^?1 for positively or negatively charged contacts. The high frequency and strength, despite variation in structure and composition, of these π-interactions point to an important function in biological systems.     

Dynamic regulation of lipid–protein interactions

We review the importance of helix motions for the function of several important categories of membrane proteins and for the properties of several model molecular systems. For voltage-gated potassium or sodium channels, sliding, tilting and/or rotational movements of the S4 helix accompanied by a swapping of cognate side-chain ion-pair interactions regulate the channel gating. In the seven-helix G protein-coupled receptors, exemplified by the rhodopsins, collective helix motions serve to activate the functional signaling. Peptides which initially associate with lipid-bilayer membrane surfaces may undergo dynamic transitions from surface-bound to tilted-transmembrane orientations, sometimes accompanied by changes in the molecularity, formation of a pore or, more generally, the activation of biological function. For single-span membrane proteins, such as the tyrosine kinases, an interplay between juxtamembrane and transmembrane domains is likely to be crucial for the regulation of dimer assembly that in turn is associated with the functional responses to external signals. Additionally, we note that experiments with designed single-span transmembrane helices offer fundamental insights into the molecular features that govern protein–lipid interactions.This article is part of a Special Issue entitled: Lipid–protein interactions.     

Assessment and significance of protein–protein interactions during development of protein biopharmaceuticals

Early development of protein biotherapeutics using recombinant DNA technology involved progress in the areas of cloning, screening, expression and recovery/purification. As the biotechnology industry matured, resulting in marketed products, a greater emphasis was placed on development of formulations and delivery systems requiring a better understanding of the chemical and physical properties of newly developed protein drugs. Biophysical techniques such as analytical ultracentrifugation, dynamic and static light scattering, and circular dichroism were used to study protein–protein interactions during various stages of development of protein therapeutics. These studies included investigation of protein self-association in many of the early development projects including analysis of highly glycosylated proteins expressed in mammalian CHO cell cultures. Assessment of protein–protein interactions during development of an IgG1 monoclonal antibody that binds to IgE were important in understanding the pharmacokinetics and dosing for this important biotherapeutic used to treat severe allergic IgE-mediated asthma. These studies were extended to the investigation of monoclonal antibody–antigen interactions in human serum using the fluorescent detection system of the analytical ultracentrifuge. Analysis by sedimentation velocity analytical ultracentrifugation was also used to investigate competitive binding to monoclonal antibody targets. Recent development of high concentration protein formulations for subcutaneous administration of therapeutics posed challenges, which resulted in the use of dynamic and static light scattering, and preparative analytical ultracentrifugation to understand the self-association and rheological properties of concentrated monoclonal antibody solutions.     

A simple method for monitoring protein–DNA interactions

A simple, efficient and cheap method is reported for monitoring interactions between single stranded desoxyribonucleic acids and proteins, using fluorescence spectroscopy and complexes of 5′-dye–DNA conjugates with bovine serum albumin as probes. In the presence of a single stranded DNA-binding protein the complexes with bovine serum albumin are disrupted, which results in a reduction of fluorescence intensity.     

Peptides and peptidomimetics as regulators of protein–protein interactions

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Alternative modulation of protein–protein interactions by small molecules

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Automated feature engineering improves prediction of protein–protein interactions

Amino Acids - Over the last decade, various machine learning (ML) and statistical approaches for protein–protein interaction (PPI) predictions have been developed to help annotating...