Protein NMR spectoscopy

The increasing resistance of many microbial strains to antibiotics, delayed laboratory results, and side effects of many chemotherapeutics has raised the need to search for sensitive diagnostics and new pro-phylactic strategies especially prevention by vaccination. Understanding the epitope/antibody interaction is the key to constructing potent vaccines and effective diagnostics. B-cell epitope mapping is a promising approach to identifying the main antigenic determinants of microorganisms, in special concern the discontinuous conformational ones. Epitope-based vaccines have remarkable privilege over the conventional ones since they are specific, able to avoid undesirable immune responses, generate long lasting immunity, and are reasonably cheaper. This up-to-date review discusses and compares the different physical, computational, and molecular methods that have been used in epitope mapping. The role of each method in the identification of potent epitopes in viruses, bacteria, fungi, parasites, as well as human diseases are tagged and documented. Simultaneously, frequent combinatorial methods are highlighted. The article aims to assist researchers to design the most suitable protocol for mapping their B-cell epitopes.

Studying disease models at the molecular level is vital for drug development in order to improve treatment and prevent a wide range of human pathologies. Microbial infections are still a major challenge because pathogens rapidly and continually evolve developing drug resistance. Cancer cells also change genetically, and current therapeutic techniques may be (or may become) ineffective in many cases. The pathology of many neurological diseases remains an enigma, and the exact etiology and underlying mechanisms are still largely unknown. Viral infections spread and develop much more quickly than does the corresponding research needed to prevent and combat these infections; the present and most relevant outbreak of SARS-CoV-2, which originated in Wuhan, China, illustrates the critical and immediate need to improve drug design and development techniques. Modern day drug discovery is a time-consuming, expensive process. Each new drug takes in excess of 10 years to develop and costs on average more than a billion US dollars. This demonstrates the need of a complete redesign or novel strategies. Nuclear Magnetic Resonance (NMR) has played a critical role in drug discovery ever since its introduction several decades ago. In just three decades, NMR has become a "gold standard" platform technology in medical and pharmacology studies. In this review, we present the major applications of NMR spectroscopy in medical drug discovery and development. The basic concepts, theories, and applications of the most commonly used NMR techniques are presented. We also summarize the advantages and limitations of the primary NMR methods in drug development.

Fluorinated amino acids serve as valuable biological probes, by reporting on local protein structure and dynamics through 19 F NMR chemical shifts. 2-fluorohistidine and 4-fluorohistidine, studied here with DFT methods, have even more capabilities for biophysical studies, as their altered pK a values, relative to histidine, allow for studies of the role of proton transfer and tautomeric state in enzymatic mechanisms. Considering the two tautomeric forms of histidine, it was found that 2-fluorohistidine primarily forms the common (for histidine) τ-tautomer at neutral pH, while 4-fluorohistidine exclusively forms the less common π-tautomer. This suggests the two isomers of fluorohistidine can also serve as probes of tautomeric form within biomolecules, both by monitoring NMR chemical shifts and by potential perturbation of the tautomeric equilibrium within biomolecules. Fluorine also enables assignment of tautomeric states in crystal structures. The differences in experimental pK a values between the isomers was found to arise from solvation effects, providing insight into the polarization and molecular properties of each isomer. Results also encompass 13 C and 19 F NMR chemical shifts, from both tautomers of 2-fluorohistidine and 4-fluorohistidine in a number of different environments. This work can serve as a guide for interpretation of spectroscopic results in biophysical studies employing 2-fluorohistidine and 4-fluorohistidine. Histidine is one of the most chemically versatile amino acids, with the neutral imidazole ring having the potential to act as Lewis acid, Lewis base, hydrogen bond donor, and hydrogen bond acceptor. Further adding to its chemical diversity is the fact that the imidazole side chain of histidine can exist in two tautomeric states: the τ (N3-H or N ε2-H) and π (N1-H or N δ1-H) tautomers, as shown in Fig. 1. Likely as a consequence of this chemical " flexibility " , histidine is present in ~50% of enzyme active sites 1. The pK a (~6) of the imidazole ring in histidine is close to physiological pH, which makes it an important amino acid in protein function 2–5. The fluorinated analogues of histidine, which exist as two different regioisomers, have dramatically lower pK a values than canonical histidine: ~1 (aqueous) for 2-fluorohistidine (2-FHis) and ~2 (aqueous) to > 3 (proteins) for 4-fluorohistidine (4-FHis). This paper focuses on the properties of 2-FHis and 4-FHis, shown in both tautomeric forms in Fig. 1, including the origin of the differences in pK a between the two regioisomers. It also serves as a guide for use and spectroscopic interpretation of fluorohistidine in biophysical studies. Multiple methods for the synthesis of fluorohistidines have been developed 6–10. The ability to incorporate these analogues of histidine into proteins has facilitated mechanistic studies of proton transfer (discussed below) and characterization of protein structure using 19 F-NMR 11–20. Interestingly, the fluorine chemical shifts of 2-and 4-fluorohistidine exhibit anomalous pH-dependencies: 2-FHis is deshielded upon protonation, while the 19 F in 4-FHis becomes more shielded at low pH 21. We recently used electronic structure calculations to show that electron delocalization/localization appears to give rise to the differences in 19 F shielding 22 .

The transcriptional activity of human estrogen receptor ERα is modulated by a number of coregulatory proteins among which calmodulin (CaM). Segment 295-311 in the hinge region of ERα has previously been proposed to be the CaM binding site. In this work, we investigate the molecular mechanism of the interaction of CaM with peptides derived from the hinge region of ERα, using a biophysical approach combining isothermal titration calorimetry, fluorescence, CD and NMR. The ERα17p peptide, corresponding to the previously identified 295-311 region of ERα, recruits mainly the C-terminal domain of Ca(4)CaM, as shown by NMR spectroscopy. In contrast, a longer peptide, ERα25p, extended on the N-terminal side (residues 287-311) interacts with both N- and C-terminal domains of Ca(4)CaM. These results lead to a new delineation of the CaM binding site, encompassing residues 287-294. In particular, fluorescence spectroscopy reveals that the conserved W(292) residue is engaged within hydrophobic pockets on Ca(4)CaM. ITC results show that ERα25p binds Ca(4)CaM with an atypical 2:1 stoichiometry and a dissociation constant in the micromolar range. Based on the NMR titration of Ca(4)CaM by ERα25p showing a biphasic behavior for several residues, we suggest that concerted conformational changes of CaM domains may be required to accommodate the binding of a second peptide. CD spectra indicate that ERα25p partially folds into an α-helix upon binding to Ca(4)CaM. Hence, ERα25p is a new CaM-binding ligand that could be appropriate for the synthesis of derivatives able to control ER-dependent transcription, particularly in the context of hormone-dependent breast tumors.