Postcard: #44 An Old Timer

This black and white photographic postcard features a man standing with a horse. He wears a cowboy hat, scarf, shirt, vest and pants. He has leather gloves with a star on them and a gun belt with a gun on the left hip. The horse to his right was a saddle, rope and cloth material tied behind the saddle. Written text is at the bottom of the card. Handwriting is on the back of the card. (Note indicates the photo was taken by Liberal, Kansas photographer Hal Reid.)https://scholars.fhsu.edu/tj_postcards/2349/thumbnail.jp

Choosing the optimal spectroscopic toolkit to understand protein function

Spectroscopy was one of the earliest methods used to study the properties and reactions of proteins, and remains one of the most powerful and widely used approaches to this day. A sometimes bewildering range of spectroscopies is now available, applicable to different sample states, timescales and indeed biological questions. This editorial describes some of the most relevant spectroscopic methods together with a selection of illustrative examples.</jats:p

Current perspectives in fragment based lead discovery (FBLD)

It is over 20 years since the first fragment-based discovery projects were disclosed. The methods are now mature for most ‘conventional’ targets in drug discovery such as enzymes (kinases and proteases) but there has also been growing success on more challenging targets, such as disruption of protein–protein interactions. The main application is to identify tractable chemical startpoints that non-covalently modulate the activity of a biological molecule. In this essay, we overview current practice in the methods and discuss how they have had an impact in lead discovery – generating a large number of fragment-derived compounds that are in clinical trials and two medicines treating patients. In addition, we discuss some of the more recent applications of the methods in chemical biology – providing chemical tools to investigate biological molecules, mechanisms and systems

Molecular dynamics studies on the NMR and X-ray structures of rabbit prion protein wild-type and mutants

Prion diseases are invariably fatal and highly infectious neurodegenerative diseases that affect a wide variety of mammalian species such as sheep, goats, mice, humans, chimpanzees, hamsters, cattle, elks, deer, minks, cats, chicken, pigs, turtles, etc. These neurodegenerative diseases are caused by the conversion from a soluble normal cellular protein into insoluble abnormally folded infectious prions and the conversion is believed to involve conformational change from a predominantly alpha-helical protein to one rich in beta-sheet structure. Such conformational changes may be amenable to study by molecular dynamics (MD) techniques. For rabbits, classical studies show they have a low susceptibility to be infected, but in 2012 it was reported that rabbit prion can be generated (though not directly) and the rabbit prion is infectious and transmissible (Proceedings of the National Academy of Sciences USA 109(13): 5080-5). This paper studies the NMR and X-ray molecular structures of rabbit prion protein wild-type and mutants by MD techniques, in order to understand the specific mechanism of rabbit prion protein and rabbit prions.Comment: (The 2nd version of arXiv1304.7633

Determination of Configuration and Conformation of a Reserpine Derivative with Seven Stereogenic Centers Using Molecular Dynamics with RDC‐Derived Tensorial Constraints

NMR-based determination of the configuration of complex molecules containing many stereocenters is often not possible using traditional NOE data and coupling patterns. Making use of residual dipolar couplings (RDCs), we were able to determine the relative configuration of a natural product containing seven stereocenters, including a chiral amine lacking direct RDC data. To identify the correct relative configuration out of 32 possible ones, experimental RDCs were used in three different approaches for data interpretation: by fitting experimental data based singular value decomposition (SVD) using a single alignment tensor and either (i) a single conformer or (ii) multiple conformers, or alternatively (iii) using molecular dynamics simulations with tensorial orientational constraints (MDOC). Even though in all three approaches one and the same configuration could be selected and clear discrimination between possible configurations was achieved, the experimental data was not fully satisfied by the methods based on single tensor approaches. While these two approaches are faster, only MDOC is able to fully reproduce experimental results, as the obtained conformational ensemble adequately covers the conformational space necessary to describe the molecule with inherent flexibility

Complex folding and misfolding effects of deer-specific amino acid substitutions in the β2-α2 loop of murine prion protein

The β2–α2 loop of PrPC is a key modulator of disease-associated prion protein misfolding. Amino acids that differentiate mouse (Ser169, Asn173) and deer (Asn169, Thr173) PrPC appear to confer dramatically different structural properties in this region and it has been suggested that amino acid sequences associated with structural rigidity of the loop also confer susceptibility to prion disease. Using mouse recombinant PrP, we show that mutating residue 173 from Asn to Thr alters protein stability and misfolding only subtly, whilst changing Ser to Asn at codon 169 causes instability in the protein, promotes oligomer formation and dramatically potentiates fibril formation. The doubly mutated protein exhibits more complex folding and misfolding behaviour than either single mutant, suggestive of differential effects of the β2–α2 loop sequence on both protein stability and on specific misfolding pathways. Molecular dynamics simulation of protein structure suggests a key role for the solvent accessibility of Tyr168 in promoting molecular interactions that may lead to prion protein misfolding. Thus, we conclude that ‘rigidity’ in the β2–α2 loop region of the normal conformer of PrP has less effect on misfolding than other sequence-related effects in this region

Accelerated Screening of Protein–Ligand Interactions via Parallel T2_2 -Weighted 19^{19}F-MRI

In drug discovery, ligands are sought that modulate the (mal-)function of medicinally relevant target proteins. In order to develop new drugs, typically a multitude of potential ligands are initially screened for binding and subsequently characterized for their affinity. Nuclear magnetic resonance (NMR) is a well-established and highly sensitive technology for characterizing such interactions. However, it has limited throughput, because only one sample can be measured at a time. In contrast, magnetic resonance imaging (MRI) is inherently parallel and MR parameters can conveniently be encoded in its images, potentially offering increased sample throughput. We explore this application using a custom-built 9-fold sample holder and a 19F-MRI coil. With this setup, we show that ligand binding can be detected by T2-weighted 19F-MRI using 4-(trifluoromethyl)benzamidine (TFBA) and trypsin as the reporter ligand and target protein, respectively. Furthermore, we demonstrate that the affinity of nonfluorinated ligands can be determined in a competition format by monitoring the dose-dependent displacement of TFBA. By comparing 19F-T2-weighted MR images of TFBA in the presence of different benzamidine (BA) concentrations–all recorded in parallel–the affinity of BA could be derived. Therefore, this approach promises parallel characterization of protein–ligand interactions and increased throughput of biochemical assays, with potential for increased sensitivity when combined with hyperpolarization techniques

Bullet-DNP Enables NMR Spectroscopy of Pyruvate and Amino Acids at Nanomolar to Low Micromolar Concentrations

Hyperpolarized pyruvate is a widely used marker to track metabolism in vivo and a benchmark molecule for hyperpolarization methods. Here, we show how a combination of improved bullet-DNP instrumentation, an optimized sample preparation and a further sensitivity increase via a$^{13}$C–$^1$H polarization transfer after dissolution enable the observation of pyruvate at a concentration of 250 nM immediately after dissolution. At this concentration, the experiment employs a total mass of pyruvate of only 20 ng or 180 pmol. If the concentration is increased to 45 μM, pyruvate may be detected 1 min after dissolution with a signal-to-noise ratio exceeding 50. The procedure can be extended to observe a mixture of amino acids at low micromolar concentrations

Macrocyclization of backbone N -methylated peptides by a prolyl oligopeptidase with a distinctive substrate recognition mechanism †

Macrocyclization and multiple backbone N-methylations can significantly improve the pharmacological properties of peptides. Since chemical synthesis of such compounds is often challenging, enzyme-based production platforms are an interesting option. Here, we characterized OphP, a serine peptidase involved in the cyclization of omphalotins, a group of ribosomally produced dodecapeptides with multiple backbone N-methylations. OphP displays robust peptidase and macrocyclase activity towards multiply α-N-methylated peptides of various lengths and composition derived from the omphalotin precursor protein OphMA. In addition, OphP processes, with lower efficiency, peptides unrelated to OphMA, containing a MeGly, MeAla or Pro residue at the P1 site. Structural analysis reveals that OphP adopts a canonical prolyl oligopeptidase fold but, unlike other enzymes of this enzyme family, recognizes its substrates by their hydrophobic and multiply backbone N-methylated core rather than by the follower peptide. The activity of OphP could be harnessed for the enzymatic production of therapeutic peptides