INVESTIGADORES
DELFINO jose maria
artículos
Título:
Recognition between a short unstructured peptide and a partially folded fragment leads to the thioredoxin fold sharing native-like dynamics
Autor/es:
BINOLFI A; FERNANDEZ CO; SICA MP; DELFINO JM; SANTOS J
Revista:
PROTEINS: STRUCTURE, FUNCTION AND GENETICS
Editorial:
WILEY-LISS, DIV JOHN WILEY & SONS INC
Referencias:
Lugar: New York; Año: 2012 vol. 80 p. 1448 - 1464
ISSN:
0887-3585
Resumen:
Thioredoxins (TRXs) constitute attractive alpha/beta scaffolds for
investigating molecular recognition. The interaction
between the recombinant fragment spanning the sequence
193 of full-length TRX (TRX1-93) and the synthetic peptide
comprising residues 94108 (TRX94-108), plus a C-terminal
tyrosine tag (the numbering scheme used in entry
pdb 2TRX is used throughout the article, two complementary
moieties of E. coli TRX, brings about the consolidation
of a native-like complex. Despite its reduced thermodynamic
stability, this complex is able to acquire fine structural
features remarkably similar to those characteristic of
full-length TRX, namely, hydrodynamic behavior, assessed
by diffusion-ordered spectroscopy (DOSY)-NMR; the pattern
of secondary structure, as revealed by three-bond
HNHa coupling constants and secondary shifts for Ha/CO/
Ca/Cb; native-like tertiary structural signatures revealed by
near-UV circular dichroism (CD) spectroscopy. The complex
exhibits a relaxation behavior compatible with that
expected for a native-like structure. However, heteronuclear
nuclear Overhauser effect (NOE)s reveal an enhanced dynamics
for the complex by comparison with full-length
TRX. Furthermore, higher R2 values for residues 4350 and
7489 would likely result from an exchange process modulated
by the peptide at the interface region. The slow kinetics
of the consolidation reaction was followed by CD and realtime
NMR. Equilibrium titration experiments by NMR yield
a KD value of 1.4 microM and a second low-affinity (>150
microM) binding event in the vicinity of the active site. Molecular
dynamics simulations of both the isolated fragment TRX1-93
and the complex suggest the destabilization of alpha2 and alpha3 helical
elements and the persistence of beta-structure in the absence
of TRX94-108. Altogether, structural and dynamic evidence
presented herein points to the key role played by the
C-terminal helix in establishing the overall fold. This critical
switch module endows reduced TRX with the ability to act as
a cooperative folding unit.