Gary López

Publications (2)11.88 Total impact

• Article: On the NMR analysis of pKa values in the unfolded state of proteins by extrapolation to zero denaturant.

  Jeniffer Quijada, Gary López, Rodney Versace, Luis Ramírez, María Luisa Tasayco
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  ABSTRACT: Detailed knowledge of the pH-dependence in both folded and unfolded states of proteins is essential to understand the role of electrostatics in protein stability. The increasing number of natively disordered proteins constitutes an excellent source for the NMR analysis of pKa values in the unfolded state of proteins. However, the tendency of many natively disordered proteins to aggregate via intermolecular hydrophobic clusters limits their NMR analysis over a wide pH range. To assess whether the pKa values in natively disordered polypeptides can be extrapolated from NMR measurements in the presence of denaturants, the natively disordered backbone of the C-terminal fragment 75 to 105 of Human Thioredoxin was studied. First, assignments using triple resonance experiments were performed to confirm lack of secondary structure. Then the pH-dependence of the amides and carboxylate side chains of Glu residues (Glu88, Glu95, Glu98, and Glu103) in the pH range from 2.0 to 7.0 was monitored using 2D 1H15N HSQC and 3D C(CO)NH experiments, and the behavior of their amides and corresponding carboxyl groups was compared to confirm the absence of nonlocal interactions. Lastly, the effect of increasing dimethyl urea concentration on the pKa values of these Glu residues was monitored. The results indicate that: (i) the dispersion in the pKa of carboxyl groups and the pH midpoints of amides in Glu residues is about 0.5 pH units and 0.6 pH units, respectively; (ii) the backbone amides of the Glu residues exhibit pH midpoints which are within 0.2 pH units from those of their carboxylates; (iii) the addition of denaturant produces upshifts in the pKa values of Glu residues that are nearly independent of their position in the sequence; and (iv) these upshifts show a nonlinear behavior in denaturant concentration, complicating the extrapolation to zero denaturant. Nevertheless, the relative ordering of the pKa values of Glu residues is preserved over the whole range of denaturant concentrations indicating that measurements at high denaturant concentration (e.g. 4 M dimethyl urea) can yield a qualitatively correct ranking of the pKa of these residues in natively disordered proteins whose pH-dependence cannot be monitored directly by NMR.
  Biophysical Chemistry 10/2007; 129(2-3):242-50. · 2.20 Impact Factor
• Source

  Available from: Damon Huber

  Article: A selection for mutants that interfere with folding of Escherichia coli thioredoxin-1 in vivo.

  Damon Huber, Myoung-Il Cha, Laurent Debarbieux, Anne-Gaëlle Planson, Nelly Cruz, Gary López, María Luisa Tasayco, Alain Chaffotte, Jon Beckwith
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  ABSTRACT: Escherichia coli thioredoxin is normally a cytoplasmic protein involved in the reduction of disulfide bonds. However, thioredoxin can be translocated to the periplasm when it is attached to a cotranslational signal sequence. When exported to the periplasm, it can partially replace the activity of DsbA in promoting the formation of disulfide bonds. In contrast, when thioredoxin is fused to a posttranslational signal sequence, very little of it appears in the periplasm. We propose that this absence of posttranslational export is due to the rapid folding of thioredoxin in the cytoplasm. We sought mutants of thioredoxin that retarded its folding in the cytoplasm, which we accomplished by fusing thioredoxin to a posttranslational signal sequence and selecting for mutants in which thioredoxin was exported to the periplasm, where it could replace DsbA. The collection of mutants obtained represents a limited number of amino acid changes in the protein. In vitro studies on purified mutant proteins show that all but one are defective in the kinetics and thermodynamics of protein folding. We propose that the slower folding of the thioredoxin mutant proteins in the cytoplasm allows their export by a posttranslational pathway. We discuss some implications of this class of mutants for aspects of the folding pathway of thioredoxin and for its mechanism of export. In particular, the finding that a folding mutant that allows protein translocation alters an amino acid at the C terminus of the protein suggests that the degree to which thioredoxin folds during its translation must be severely restricted.
  Proceedings of the National Academy of Sciences 01/2006; 102(52):18872-7. · 9.68 Impact Factor