12 research outputs found
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
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
Experimental characterization of disordered and ordered aggregates populated during the process of amyloid fibril formation.
Recent experimental evidence points to intermediates populated during the process of amyloid fibril formation as the toxic moieties primarily responsible for the development of increasingly common disorders such as Alzheimer's disease and type II diabetes. We describe here the application of a pulse-labeling hydrogen-deuterium (HD) exchange strategy monitored by mass spectrometry (MS) and NMR spectroscopy (NMR) to characterize the aggregation process of an SH3 domain under 2 different conditions, both of which ultimately lead to well-defined amyloid fibrils. Under one condition, the intermediates appear to be largely amorphous in nature, whereas under the other condition protofibrillar species are clearly evident. Under the conditions favoring amorphous-like intermediates, only species having no protection against HD exchange can be detected in addition to the mature fibrils that show a high degree of protection. By contrast, under the conditions favoring protofibrillar-like intermediates, MS reveals that multiple species are present with different degrees of HD exchange protection, indicating that aggregation occurs initially through relatively disordered species that subsequently evolve to form ordered aggregates that eventually lead to amyloid fibrils. Further analysis using NMR provides residue-specific information on the structural reorganizations that take place during aggregation, as well as on the time scales by which they occur
Letter to the Editor: 1H(N), 15N, 13CO, 13Cα, 13Cβ assignment and secondary structure of a 20 kDa α-l-fucosidase from pea using TROSY
This work was supported by grants BIO99-484 from ComisiĂłn de InvestigaciĂłn CientĂfica y TĂ©cnica, Madrid (Spain) and Generalitat de Catalunya [Grup Consolidat (1999SGR0042) i Centre de Referència de Biotecnologia].Peer reviewe
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
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
Peptide affinity chromatography based on combinatorial strategies for protein purification
We describe a method to develop affinity chromatography matrices with short peptide ligands for protein purification. The method entitles the following: a) synthesis of a combinatorial library on the HMBA-ChemMatrix resin by the divide-couple-recombine (DCR) method using the Fmoc chemistry, b) library screening with the protein of interest labeled with a fluorescent dye or biotin, c) identification of peptides contained on positive beads by tandem matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS/MS), d) solid phase peptide ligands synthesis and immobilization in chromatographic supports, e) evaluation of protein adsorption on peptide affinity matrices from the equilibrium isotherms and breakthrough curves.Fil: Camperi, Silvia Andrea. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y BioquĂmica. Departamento de MicrobiologĂa, InmunologĂa y BiotecnologĂa. Cátedra de MicrobiologĂa Industrial y BiotecnologĂa; ArgentinaFil: MartĂnez Ceron, MarĂa Camila. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y BioquĂmica. Departamento de MicrobiologĂa, InmunologĂa y BiotecnologĂa. Cátedra de MicrobiologĂa Industrial y BiotecnologĂa; ArgentinaFil: Giudicessi, Silvana Laura. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y BioquĂmica. Departamento de MicrobiologĂa, InmunologĂa y BiotecnologĂa. Cátedra de MicrobiologĂa Industrial y BiotecnologĂa; ArgentinaFil: Marani, Mariela Mirta. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y BioquĂmica. Departamento de MicrobiologĂa, InmunologĂa y BiotecnologĂa. Cátedra de MicrobiologĂa Industrial y BiotecnologĂa; ArgentinaFil: Albericio Palomera, Fernando. Universidad de Barcelona; EspañaFil: Cascone, Osvaldo. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y BioquĂmica. Departamento de MicrobiologĂa, InmunologĂa y BiotecnologĂa. Cátedra de MicrobiologĂa Industrial y BiotecnologĂa; Argentin
Molecular recycling within amyloid fibrils.
Amyloid fibrils are thread-like protein aggregates with a core region formed from repetitive arrays of beta-sheets oriented parallel to the fibril axis. Such structures were first recognized in clinical disorders, but more recently have also been linked to a variety of non-pathogenic phenomena ranging from the transfer of genetic information to synaptic changes associated with memory. The observation that many proteins can convert into similar structures in vitro has suggested that this ability is a generic feature of polypeptide chains. Here we have probed the nature of the amyloid structure by monitoring hydrogen/deuterium exchange in fibrils formed from an SH3 domain using a combination of nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry. The results reveal that under the conditions used in this study, exchange is dominated by a mechanism of dissociation and re-association that results in the recycling of molecules within the fibril population. This insight into the dynamic nature of amyloid fibrils, and the ability to determine the parameters that define this behaviour, have important implications for the design of therapeutic strategies directed against amyloid disease