Exclusively Heteronuclear 13C‐Detected Amino‐Acid‐Selective NMR Experiments for the Study of Intrinsically Disordered Proteins (IDPs)

Carbon‐13 direct‐detection NMR methods have proved to be very useful for the characterization of intrinsically disordered proteins (IDPs). Here we present a suite of experiments in which amino‐acid‐selective editing blocks are encoded in CACON‐ and CANCO‐type sequences to give ¹³C‐detected spectra containing correlations arising from a particular type or group of amino acid(s). These two general types of experiments provide the complementary intra‐ and inter‐residue correlations necessary for sequence‐specific assignment of backbone resonance frequencies. We demonstrate the capabilities of these experiments on two IDPs: fully reduced Cox17 and WIP^C. The proposed approach constitutes an independent strategy to simplify crowded spectra as well as to perform sequence‐specific assignment, thereby demonstrating its potential to study IDPs.     

Multi‐phosphorylation of the Intrinsically Disordered Unique Domain of c‐Src Studied by In‐Cell and Real‐Time NMR Spectroscopy

Intrinsically disordered regions (IDRs) are preferred sites for post‐translational modifications essential for regulating protein function. The enhanced local mobility of IDRs facilitates their observation by NMR spectroscopy in vivo. Phosphorylation events can occur at multiple sites and respond dynamically to changes in kinase–phosphatase networks. Here we used real‐time NMR spectroscopy to study the effect of kinases and phosphatases present in Xenopus oocytes and egg extracts on the phosphorylation state of the “unique domain” of c‐Src. We followed the phosphorylation of S17 in oocytes, and of S17, S69, and S75 in egg extracts by NMR spectroscopy, MS, and western blotting. Addition of specific kinase inhibitors showed that S75 and S69 are phosphorylated by CDKs (cyclin‐dependent kinases) differently from Cdk1. Moreover, although PKA (cAMP‐dependent protein kinase) can phosphorylate S17 in vitro, this was not the major S17 kinase in egg extracts. Changes in PKA activity affected the phosphorylation levels of CDK‐dependent sites, thus suggesting indirect effects of kinase–phosphatase networks. This study provides a proof‐of‐concept of the use of real‐time in vivo NMR spectroscopy to characterize kinase/phosphatase effects on intrinsically disordered regulatory domains.     

Protein Regulation by Intrinsically Disordered Regions: A Role for Subdomains in the IDR of the HIV‐1 Rev Protein

Intrinsically disordered regions (IDRs) in proteins are highly abundant, but they are still commonly viewed as long stretches of polar, solvent‐accessible residues. Here we show that the disordered C‐terminal domain (CTD) of HIV‐1 Rev has two subregions that carry out two distinct complementary roles of regulating protein oligomerization and contributing to stability. We propose that this takes place through a delicate balance between charged and hydrophobic residues within the IDR. This means that mutations in this region, as well as the known mutations in the structured region of the protein, can affect protein function. We suggest that IDRs in proteins should be divided into subdomains similarly to structured regions, rather than being viewed as long flexible stretches.     

Leucine Side‐Chain Conformation and Dynamics in Proteins from 13C NMR Chemical Shifts

Look to the left : The carbon nucleus of a substituent in the gauche position about a subtending dihedral angle experiences an NMR chemical shift of about 5 ppm relative to the same chemical group present in the trans position. We demonstrate that this “γ‐gauche effect” can be utilized to determine the conformation and extent of rotameric averaging for leucine amino acid side chains in the protein calbindin D_9k. The success of this approach suggests that rules can be established to define the orientation of other side chains in proteins as well, offering an easy gauge of protein side‐chain flexibility, as well as avenues to advance protein structure determination by using side‐chain chemical shifts.

     

NMR Characterization of Long-Range Contacts in Intrinsically Disordered Proteins from Paramagnetic Relaxation Enhancement in 13C Direct-Detection Experiments

Intrinsically disordered proteins (IDPs) carry out many biological functions. They lack a stable 3D structure and are able to adopt many different conformations in dynamic equilibrium. The interplay between local dynamics and global rearrangements is key for their function. A widely used experimental NMR spectroscopy approach to study long-range contacts in IDPs exploits paramagnetic effects, and ¹H detection experiments are generally used to determine paramagnetic relaxation enhancement (PRE) for amide protons. However, under physiological conditions, exchange broadening hampers the detection of solvent-exposed amide protons, which reduces the content of information available. Herein, we present an experimental approach based on direct carbon detection of PRE that provides improved resolution, reduced sensitivity to exchange broadening, and complementary information derived from the use of different starting polarization sources.     

The Casein Kinase 2‐Dependent Phosphorylation of NS5A Domain 3 from Hepatitis C Virus Followed by Time‐Resolved NMR Spectroscopy

Hepatitis C virus (HCV) chronically affects millions of individuals worldwide. The HCV nonstructural protein 5A (NS5A) plays a critical role in the viral assembly pathway. Domain 3 (D3) of NS5A is an unstructured polypeptide responsible for the interaction with the core particle assembly structure. Casein kinase 2 (CK2) phosphorylates NS5A‐D3 at multiple sites that have mostly been predicted and only observed indirectly. In order to identify the CK2‐dependent phosphorylation sites, we monitored the reaction between NS5A‐D3 and CK2 in vitro by time‐resolved NMR. We unambiguously identified four serine residues as substrates of CK2. The apparent rate constant for each site was determined from the reaction curves. Ser408 was quickly phosphorylated, whereas the three other serine residues reacted more slowly. These results provide a starting point from which to elucidate the role of phosphorylation in the mechanisms of viral assembly—and in the modulation of the viral activity—at the molecular level.     

N‐Lauroylation during the Expression of Recombinant N‐Myristoylated Proteins: Implications and Solutions

Incorporation of myristic acid onto the N terminus of a protein is a crucial modification that promotes membrane binding and correct localization of important components of signaling pathways. Recombinant expression of N‐myristoylated proteins in Escherichia coli can be achieved by co‐expressing yeast N‐myristoyltransferase and supplementing the growth medium with myristic acid. However, undesired incorporation of the 12‐carbon fatty acid lauric acid can also occur (leading to heterogeneous samples), especially when the available carbon sources are scarce, as it is the case in minimal medium for the expression of isotopically enriched samples. By applying this method to the brain acid soluble protein 1 and the 1–185 N‐terminal region of c‐Src, we show the significant, and protein‐specific, differences in the membrane binding properties of lauroylated and myristoylated forms. We also present a robust strategy for obtaining lauryl‐free samples of myristoylated proteins in both rich and minimal media.     

Fluorine NMR Spectroscopy Enables to Quantify the Affinity Between DNA and Proteins in Cell Lysate

The determination of the binding affinity quantifying the interaction between proteins and nucleic acids is of crucial interest in biological and chemical research. Here, we have made use of site-specific fluorine labeling of the cold shock protein from Bacillus subtilis, BsCspB, enabling to directly monitor the interaction with single stranded DNA molecules in cell lysate. High-resolution ¹⁹F NMR spectroscopy has been applied to exclusively report on resonance signals arising from the protein under study. We have found that this experimental approach advances the reliable determination of the binding affinity between single stranded DNA molecules and its target protein in this complex biological environment by intertwining analyses based on NMR chemical shifts, signal heights, line shapes and simulations. We propose that the developed experimental platform offers a potent approach for the identification of binding affinities characterizing intermolecular interactions in native surroundings covering the nano-to-micromolar range that can be even expanded to in cell applications in future studies.     

Impact of Hydrostatic Pressure on an Intrinsically Disordered Protein: A High‐Pressure NMR Study of α‐Synuclein

The impact of pressure on the backbone ¹⁵N, ¹H and ¹³C chemical shifts in N‐terminally acetylated α‐synuclein has been evaluated over a pressure range 1–2500 bar. Even while the chemical shifts fall very close to random coil values, as expected for an intrinsically disordered protein, substantial deviations in the pressure dependence of the chemical shifts are seen relative to those in short model peptides. In particular, the nonlinear pressure response of the ¹H^N chemical shifts, which commonly is associated with the presence of low‐lying “excited states”, is much larger in α‐synuclein than in model peptides. The linear pressure response of ¹H^N chemical shift, commonly linked to H‐bond length change, correlates well with those in short model peptides, and is found to be anticorrelated with its temperature dependence. The pressure dependence of ¹³C chemical shifts shows remarkably large variations, even when accounting for residue type, and do not point to a clear shift in population between different regions of the Ramachandran map. However, a nearly universal decrease in ³J_HN–Hα by 0.22±0.05 Hz suggests a slight increase in population of the polyproline II region at 2500 bar. The first six residues of N‐terminally acetylated synuclein show a transient of approximately 15 % population of α‐helix, which slightly diminishes at 2500 bar. The backbone dynamics of the protein is not visibly affected beyond the effect of slight increase in water viscosity at 2500 bar.     

Substrate Binding Drives Active‐Site Closing of Human Blood Group B Galactosyltransferase as Revealed by Hot‐Spot Labeling and NMR Spectroscopy Experiments

Crystallography has shown that human blood group A (GTA) and B (GTB) glycosyltransferases undergo transitions between “open”, “semiclosed”, and “closed” conformations upon substrate binding. However, the timescales of the corresponding conformational reorientations are unknown. Crystal structures show that the Trp and Met residues are located at “conformational hot spots” of the enzymes. Therefore, we utilized ¹⁵N side‐chain labeling of Trp residues and ¹³C‐methyl labeling of Met residues to study substrate‐induced conformational transitions of GTB. Chemical‐shift perturbations (CSPs) of Met and Trp residues in direct contact with substrate ligands reflect binding kinetics, whereas the CSPs of Met and Trp residues at remote sites reflect conformational changes of the enzyme upon substrate binding. Acceptor binding is fast on the chemical‐shift timescale with rather small CSPs in the range of less than approximately 20 Hz. Donor binding matches the intermediate exchange regime to yield an estimate for exchange rate constants of approximately 200–300 Hz. Donor or acceptor binding to GTB saturated with acceptor or donor substrate, respectively, is slow (<10 Hz), as are coupled protein motions, reflecting mutual allosteric control of donor and acceptor binding. Remote CSPs suggest that substrate binding drives the enzyme into the closed state required for catalysis. These findings should contribute to better understanding of the mechanism of glycosyl transfer of GTA and GTB.     

Unambiguous Assignment of Short‐ and Long‐Range Structural Restraints by Solid‐State NMR Spectroscopy with Segmental Isotope Labeling

We present an efficient method for the reduction of spectral complexity in the solid‐state NMR spectra of insoluble protein assemblies, without loss of signal intensity. The approach is based on segmental isotope labeling by using the split intein DnaE from Nostoc punctiforme. We show that the segmentally ¹³C,¹⁵N‐labeled prion domain of HET‐s exhibits significantly reduced spectral overlap while retaining the wild‐type structure and spectral quality. A large number of unambiguous distance restraints were thus collected from a single two‐dimensional ¹³C,¹³C cross‐correlation spectrum. The observed resonances could be unambiguously identified as intramolecular without the need for preparing a dilute, less sensitive sample.     

Solution‐NMR Characterization of Outer‐Membrane Protein A from E. coli in Lipid Bilayer Nanodiscs and Detergent Micelles

X‐ray crystallography and solution NMR of detergent‐reconstituted OmpA (outer membrane protein A from E. coli) had shown that this protein forms an eight‐stranded transmembrane β‐barrel, but only limited information was obtained for the extracellular loops. In NMR studies of OmpA in two different detergent micelles, “NMR‐invisible” amino acid residues in‐between the extracellular loops and the β‐barrel prevented complete structural characterization. Here, we show that this NMR‐invisible ring around the β‐barrel of OmpA is also present in lipid bilayer nanodiscs and in mixed micelles with a third detergent, thus suggesting that the implicated rate processes have a functional role rather than representing an artifact of the protein reconstitution. In addition to sequence‐specific NMR assignments for OmpA in the nanodiscs, the present results are based on a protocol of micro‐coil TROSY‐ and CRINEPT‐type NMR diffusion measurements for studying the hydrodynamic properties and the foldedness of [²H,¹⁵N]‐labeled membrane proteins in nanodiscs. This protocol can be applied under conditions closely similar to those used for NMR structure determinations or crystallization trials.     

Impact of the C‐terminal Disulfide Bond on the Folding and Stability of Onconase

The two homologous proteins ribonuclease A and onconase fold through conserved initial contacts but differ significantly in their thermodynamic stability. A disulfide bond is located in the folding initiation site of onconase (the C‐terminal part of the protein molecule) that is missing in ribonuclease A, whereas the other three disulfide bonds of onconase are conserved in ribonuclease A. Consequently, the deletion of this C‐terminal disulfide bond (C87–C104) allows the impact of the contacts in this region on the folding of onconase to be studied. We found the C87A/C104A‐onconase variant to be less active and less stable than the wild‐type protein, whereas the tertiary structure, which was determined by both X‐ray crystallography and NMR spectroscopy, was only marginally affected. The folding kinetics of the variant, however, were found to be changed considerably in comparison to wild‐type onconase. Proton exchange experiments in combination with two‐dimensional NMR spectroscopy revealed differences in the native‐state dynamics of the two proteins in the folding initiation site, which are held responsible for the changed folding mechanism. Likewise, the molecular dynamics simulation of the unfolding reaction indicated disparities for both proteins. Our results show that the high stability of onconase is based on the efficient stabilization of the folding initiation site by the C‐terminal disulfide bond. The formation of the on‐pathway intermediate, which is detectable during the folding of the wild‐type protein and promotes the fast and efficient refolding reaction, requires the presence of this covalent bond.