High-Resolution Insight into G‑Overhang Architecture

NMR and fluorescence spectroscopy were used to address the effect of intracellular molecular crowding and related hydration on a model telomeric G-quadruplex (G4) DNA structure (d­(AG₃­(TTAGGG)₃)). d­(AG₃­(TTAGGG)₃) prevalently adopted the hybrid-1 conformation <i>in vivo</i>, <i>ex vivo</i>, and in dilute potassium-based solution, while it formed the parallel propeller fold in water-depleted potassium-based solution, a commonly used model system for studying intracellular molecular crowding. The dilute potassium-based solution appeared to imitate the properties of the cellular environment required for d­(AG₃­(TTAGGG)₃) folding under <i>in vivo</i> and <i>ex vivo</i> conditions. High-resolution NMR investigations of site-specifically ¹⁵N-labeled G4 units in native-like single-stranded telomeric DNA revealed that the 3′-terminal and internal G4 unit predominantly coexist in 2-tetrad antiparallel basket and hybrid-2 structures that are arranged in “beads-on-a-string”-like fashion. Our data provide the first high-resolution insight into the telomeric G-overhang architecture under essentially physiological conditions and identify the 2-tetrad antiparallel basket and hybrid-2 topologies as the structural targets for the development of telomere-specific G4 ligands

Deconstructing Protein Binding of Sulfonamides and Sulfonamide Analogues

Sulfonamides are one of the most important pharmacophores in medicinal chemistry, and sulfonamide analogues have gained substantial interest in recent years. However, the protein interactions of sulfonamides and especially of their analogues are underexplored. Using FKBP12 as a model system, we describe the synthesis of optically pure sulfenamide, sulfinamide, and sulfonimidamide analogues of a well characterized sulfonamide ligand. This allowed us to precisely determine the binding contributions of each sulfonamide oxygen atom and the consequences of nitrogen replacements. We also present high resolution cocrystal structures of sulfonamide analogues buried in the pocket of a protein target. This revealed intimate contacts with the protein including an unprecedented hydrogen bond acceptor of sulfonimidamides. The use of sulfonamide analogues enabled new exit vectors that allowed remodeling of a subpocket in FKBP12. Our results illuminate the protein interaction potential of sulfonamides sulfonamide analogues and will aid in their rational desig

Single-Molecule Force Spectroscopy from Nanodiscs: An Assay to Quantify Folding, Stability, and Interactions of Native Membrane Proteins

Single-molecule force spectroscopy (SMFS) can quantify and localize inter- and intramolecular interactions that determine the folding, stability, and functional state of membrane proteins. To conduct SMFS the membranes embedding the membrane proteins must be imaged and localized in a rather time-consuming manner. Toward simplifying the investigation of membrane proteins by SMFS, we reconstituted the light-driven proton pump bacteriorhodopsin into lipid nanodiscs. The advantage of using nanodiscs is that membrane proteins can be handled like water-soluble proteins and characterized with similar ease. SMFS characterization of bacteriorhodopsin in native purple membranes and in nanodiscs reveals no significant alterations of structure, function, unfolding intermediates, and strengths of inter- and intramolecular interactions. This demonstrates that lipid nanodiscs provide a unique approach for <i>in vitro</i> studies of native membrane proteins using SMFS and open an avenue to characterize membrane proteins by a wide variety of SMFS approaches that have been established on water-soluble proteins

Combining <i>in Vitro</i> Folding with Cell Free Protein Synthesis for Membrane Protein Expression

Cell free protein synthesis (CFPS) has emerged as a promising methodology for protein expression. While polypeptide production is very reliable and efficient using CFPS, the correct cotranslational folding of membrane proteins during CFPS is still a challenge. In this contribution, we describe a two-step protocol in which the integral membrane protein is initially expressed by CFPS as a precipitate followed by an <i>in vitro</i> folding procedure using lipid vesicles for converting the protein precipitate to the correctly folded protein. We demonstrate the feasibility of using this approach for the K⁺ channels KcsA and MVP and the amino acid transporter LeuT. We determine the crystal structure of the KcsA channel obtained by CFPS and <i>in vitro</i> folding to show the structural similarity to the cellular expressed KcsA channel and to establish the feasibility of using this two-step approach for membrane protein production for structural studies. Our studies show that the correct folding of these membrane proteins with complex topologies can take place <i>in vitro</i> without the involvement of the cellular machinery for membrane protein biogenesis. This indicates that the folding instructions for these complex membrane proteins are contained entirely within the protein sequence

Insights into Cotranslational Membrane Protein Insertion by Combined LILBID-Mass Spectrometry and NMR Spectroscopy

Cotranslational insertion of membrane proteins into defined nanoparticle membranes has been developed as an efficient process to produce highly soluble samples in native-like environments and to study lipid-dependent effects on protein structure and function. Numerous examples of the structural and functional characterization of transporters, ion channels, or G-protein-coupled receptors in cotranslationally formed nanodisc complexes demonstrate the versatility of this approach, although the basic underlying mechanisms of membrane insertion are mainly unknown. We have revealed the first aspects of the insertion of proteins into nanodiscs by combining cell-free expression, noncovalent mass spectrometry, and NMR spectroscopy. We provide evidence of cooperative insertion of homo-oligomeric complexes and demonstrate the possibility to modulate their stoichiometry by modifying reaction conditions. Additionally, we show that significant amounts of lipid are released from the nanodiscs upon insertion of larger protein complexes

Molecular Crowding Drives Active Pin1 into Nonspecific Complexes with Endogenous Proteins Prior to Substrate Recognition

Proteins and nucleic acids maintain the crowded interior of a living cell and can reach concentrations in the order of 200–400 g/L which affects the physicochemical parameters of the environment, such as viscosity and hydrodynamic as well as nonspecific strong repulsive and weak attractive interactions. Dynamics, structure, and activity of macromolecules were demonstrated to be affected by these parameters. However, it remains controversially debated, which of these factors are the dominant cause for the observed alterations <i>in vivo</i>. In this study we investigated the globular folded peptidyl-prolyl isomerase Pin1 in Xenopus laevis oocytes and in native-like crowded oocyte extract by in-cell NMR spectroscopy. We show that active Pin1 is driven into nonspecific weak attractive interactions with intracellular proteins prior to substrate recognition. The substrate recognition site of Pin1 performs specific and nonspecific attractive interactions. Phosphorylation of the WW domain at Ser16 by PKA abrogates both substrate recognition and the nonspecific interactions with the endogenous proteins. Our results validate the hypothesis formulated by McConkey that the majority of globular folded proteins with surface charge properties close to neutral under physiological conditions reside in macromolecular complexes with other sticky proteins due to molecular crowding. In addition, we demonstrate that commonly used synthetic crowding agents like Ficoll 70 are not suitable to mimic the intracellular environment due to their incapability to simulate biologically important weak attractive interactions

Characterization of Molecular Interactions between ACP and Halogenase Domains in the Curacin A Polyketide Synthase

Polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) are large multidomain proteins present in microorganisms that produce bioactive compounds. Curacin A is such a bioactive compound with potent anti-proliferative activity. During its biosynthesis the growing substrate is bound covalently to an acyl carrier protein (ACP) that is able to access catalytic sites of neighboring domains for chain elongation and modification. While ACP domains usually occur as monomers, the curacin A cluster codes for a triplet ACP (ACP_I-ACP_II-ACP_III) within the CurA PKS module. We have determined the structure of the isolated holo-ACP_I and show that the ACPs are independent of each other within this tridomain system. In addition, we have determined the structure of the 3-hydroxyl-3-methylglutaryl-loaded holo-ACP_I, which is the substrate for the unique halogenase (Hal) domain embedded within the CurA module. We have identified the interaction surface of both proteins using mutagenesis and MALDI-based identification of product formation. Amino acids affecting product formation are located on helices II and III of ACP_I and form a contiguous surface. Since the CurA Hal accepts substrate only when presented by one of the ACPs within the ACP_I-ACP_II-ACP_III tridomain, our data provide insight into the specificity of the chlorination reaction