The two isoforms of Lyn display different intramolecular fuzzy complexes with the SH3 domain

The function of the intrinsically disordered Unique domain of the Src family of tyrosine kinases (SFK), where the largest differences between family members are concentrated, remains poorly understood. Recent studies in c-Src have demonstrated that the Unique region forms transient interactions, described as an intramolecular fuzzy complex, with the SH3 domain and suggested that similar complexes could be formed by other SFKs. Src and Lyn are members of a distinct subfamily of SFKs. Lyn is a key player in the immunologic response and exists in two isoforms originating from alternative splicing in the Unique domain. We have used NMR to compare the intramolecular interactions in the two isoforms and found that the alternatively spliced segment interacts specifically with the so-called RT-loop in the SH3 domain and that this interaction is abolished when a polyproline ligand binds to the SH3 domain. These results support the generality of the fuzzy complex formation in distinct subfamilies of SFKs and its physiological role, as the naturally occurring alternative splicing modulates the interactions in this complex

A myristoyl binding site in the SH3 domain modulates c-Src membrane anchoring

The c-Src oncogene is anchored to the cytoplasmic membrane through its N-terminal myristoylated SH4 domain. This domain is part of an intramolecular fuzzy complex with the SH3 and Unique domains. Here we show that the N-terminal myristoyl group binds to the SH3 domain in the proximity of the RT loop, when Src is not anchored to a lipid membrane. Residues in the so-called Unique Lipid Binding Region modulate this interaction. In the presence of lipids, the myristoyl group is released from the SH3 domain and inserts into the lipid membrane. The fuzzy complex with the SH4 and Unique domains is retained in the membrane-bound form, placing the SH3 domain close to the membrane surface and restricting its orientation. The apparent affinity of myristoylated proteins containing the SH4, Unique, and SH3 domains is modulated by these intramolecular interactions, suggesting a mechanism linking c-Src activation and membrane anchoring

Active site-directed inhibitors of prolyl oligopeptidase abolishes its conformational dynamics

Deciphering conformational dynamics is crucial for understanding the biological functions of proteins and for designing compounds targeting them. In particular, providing an accurate description of microsecond-millisecond motions opens the opportunity for regulating protein-protein interactions (PPIs) by modulating the dynamics of one interacting partner. Here we analyzed the conformational dynamics of prolyl oligopeptidase (POP) and the effects of active-site-directed inhibitors on the dynamics. We used an integrated structural biology approach based on NMR spectroscopy and SAXS experiments complemented by MD simulations. We found that POP is in a slow equilibrium in solution between open and closed conformations, and that inhibitors effectively abolished this equilibrium by stabilizing the enzyme in the closed conformation

An optimized method for 15N R1 relaxation rate measurements in non-deuterated proteins

15N longitudinal relaxation rates are extensively used for the characterization of protein dynamics; however, their accurate measurement is hindered by systematic errors. 15N CSA/1H-15N dipolar cross-correlated relaxation (CC) and amide proton exchange saturation transfer from water protons are the two main sources of systematic errors in the determination of 15N R1 rates through 1H-15N HSQC-based experiments. CC is usually suppressed through a train of 180° proton pulses applied during the variable 15N relaxation period (T), which can perturb water magnetization. Thus CC cancellation is required in such a way as to minimize water saturation effects. Here we examined the level of water saturation during the T period caused by various types of inversion proton pulses to suppress CC: (I) amide-selective IBURP-2; (II) cosine-modulated IBURP-2; (III) Watergate-like blocks; and (IV) non-selective hard. We additionally demonstrate the effect of uncontrolled saturation of aliphatic protons on 15N R1 rates. In this paper we present an optimized pulse sequence that takes into account the crucial effect of controlling also the saturation of the aliphatic protons during 15N R1 measurements in non-deuterated proteins. We show that using cosine-modulated IBURP-2 pulses spaced 40 ms to cancel CC in this optimized pulse program is the method of choice to minimiz

Side chain to main chain hydrogen bonds stabilize a polyglutamine helix in a transcription factor

Polyglutamine (polyQ) tracts are low-complexity regions and their expansion is linked to certain neurodegenerative diseases. Here the authors combine experimental and computational approaches to find that the length of the androgen receptor polyQ tract correlates with its helicity and show that the polyQ helical structure is stabilized by hydrogen bonds between the Gln side chains and main chain carbonyl groups

Nuclear magnetic resonance (NMR) spectroscopy

La capacitat de la RMN per obtenir informació molecular a escala atòmica l’ha convertida en una poderosa tècnica en biomedicina i biotecnologia. Entre les seves aplicacions pot destacar-se, per exemple, la caracterització de productes naturals i sintètics i la identificació i quantificació de components individuals de barreges complexes, eines clau en les indústries farmacèutica o alimentària. A més és una tècnica molt valuosa en diagnosi mèdica. Ens permet obtenir imatges de parts del cos humà (RMN d’imatge) per detectar tumors o lesions. Possibilita la identificació de metabòlits com a possibles biomarcadors de malalties específiques. Addicionalment s’utilitza en la determinació de l’estructura tridimensional, la dinàmica i el funcionament de les principals dianes terapèutiques: les proteïnes, i també en l’estudi de les interaccions entre aquestes dianes i els fàrmacs, punt clau en el desenvolupament i millora d’un medicament.Paraules clau: qualitat alimentària, metabolòmica, estructura i dinàmica de proteïnes, disseny de fàrmacs.NMR has become a powerful technique in biomedicine and biotechnology because of its ability to provide molecular information at atomic level. Among its many applications, we highlight its use for natural products characterization and the identification and quantification of individual components of complex mixtures in the pharmaceutical or food industries. NMR is a potent tool in medical diagnosis as well. Body images obtained through nuclear magnetic resonance imaging (NMRI) inform of the presence of tumours or injuries, and metabolites can be identified as specific disease-related biomarkers. In addition, NMR provides advantages in determining the structure, dynamics and function of drug targets such as proteins, and it can characterize drug-target interactions, which are critical to the drug discovery process.Keywords: food quality control, metabolomics, protein structure anddynamics, drug discovery

Nuclear magnetic resonance

Podeu consultar el llibre complet a: http://hdl.handle.net/2445/32166This article reviews the principles and methods of nuclear magnetic resonance spectroscopy, and gives examples of applications carried out at our Facility, which illustrate the capabilities of the technique