RESUMEN
The protein encoded by the G0/G1 switch gene 2 (G0S2) is a potent inhibitor of adipose triglyceride lipase (ATGL) and thus an important regulator of intracellular lipolysis. Since dysfunction of lipolysis is associated with metabolic diseases including diabetes and obesity, inhibition of ATGL is considered a therapeutic strategy. G0S2 interacts with ATGL's patatin-domain to mediate non-competitive inhibition, however atomic details of the inhibition mechanism are incompletely understood. Sequences of G0S2 from higher organisms show a highly conserved N-terminal part, including a hydrophobic region covering amino acids 27 to 42. We show that predicted G0S2 orthologs from platypus, chicken and Japanese rice-fish are able to inhibit human and mouse ATGL, emphasizing the contribution of conserved amino acid to ATGL inhibition. Our site directed mutagenesis and truncation studies give insights in the protein-protein interaction on a per-residue level. We determine that the minimal sequence required for ATGL inhibition ranges from amino acids 20 to 44. Residues Y27, V28, G30, A34 G37, V39 or L42 within this sequence play a substantial role in ATGL inhibition. Furthermore, we show that unspecific interactions of the N-terminal part (amino acids 20-27) of the minimal sequence facilitate the interaction to ATGL. Our studies also demonstrate that full-length G0S2 shows higher tolerance to specific single amino acid exchanges in the hydrophobic region due to the stronger contributions of unspecific interactions. However, exchanges of more than one amino-acid in the hydrophobic region also result in the loss of function as ATGL inhibitor even in the full-length protein.
Asunto(s)
LipólisisAsunto(s)
Antígenos Ly/genética , Queratodermia Palmoplantar/metabolismo , Linfocitos/metabolismo , Mutación Missense/genética , Activador de Plasminógeno de Tipo Uroquinasa/genética , Antígenos Ly/química , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Proteínas Ligadas a GPI/química , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Heterogeneidad Genética , Humanos , Queratodermia Palmoplantar/genética , Unión Proteica , Activador de Plasminógeno de Tipo Uroquinasa/química , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7/metabolismoRESUMEN
Selective pulses have been used frequently for small molecules. However, their application to proteins and other macromolecules has been limited. The long duration of shaped-selective pulses and the short T(2) relaxation times in proteins often prohibited the use of highly selective pulses especially on larger biomolecules. A very selective excitation can be obtained within a short time by using the selective excitation sequence presented in this paper. Instead of using a shaped low-intensity radiofrequency pulse, a cluster of hard 90 degrees pulses, delays of free precession, and pulsed field gradients can be used to selectively excite a narrow chemical shift range within a relatively short time. Thereby, off-resonance magnetization, which is allowed to evolve freely during the free precession intervals, is destroyed by the gradient pulses. Off-resonance excitation artifacts can be removed by random variation of the interpulse delays. This leads to an excitation profile with selectivity as well as phase and relaxation behavior superior to that of commonly used shaped-selective pulses. Since the evolution of scalar coupling is inherently suppressed during the double-selective excitation of two different scalar-coupled nuclei, the presented pulse cluster is especially suited for simultaneous highly selective excitation of N-H and C-H fragments. Experimental examples are demonstrated on hen egg white lysozyme (14 kD) and the bacterial antidote ParD (19 kD).
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Matemática , Fenómenos Físicos , Física , Factores de TiempoRESUMEN
ParD is a small, acidic protein from the partitioning system of the plasmid RK2/RP4. The ParD protein exhibits specific DNA binding activity and, as the antidote component of a toxin-antidote plasmid addiction system, ParD forms a tight complex in solution with its toxin antagonist, the ParE protein. Unopposed ParE acts as a toxin that causes growth retardation and killing of plasmid cured cells. ParD negatively autoregulates its expression by binding to an operator sequence in the parDE promoter region. This DNA binding activity is crucial for the regulation of the relative abundance of toxin and antidote which ultimately determines life or death for the bacterial host and its daughter cells. In light scattering studies and gel filtration chromatography we observed the existence of a stable dimer of ParD in solution. The stoichiometry of ParD-DNA complex formation appeared to be 4:1, the molecular mass of the complex was 72.1 kDa. The alpha-helical content of ParD as determined by CD-spectrometry was 35%. The protein exhibited high thermostability with a T(M) of 64 degrees C and deltaH of 25 kcal/mol as shown by differential scanning calorimetry. Upon complex formation the T(M) increased by 10 degrees C. The thermal unfolding of the ParD protein was highly reversible as observed in repeated DSC scans of the same sample. The recovery of the native fold was proven by CD-spectroscopy.