Dynamic structure of a highly ordered beta-sheet molten globule: multiple conformations with a stable core

Area of Science:

• Protein structure and dynamics
• Biophysical chemistry
• Nuclear Magnetic Resonance (NMR) spectroscopy

Background:

• Bovine pancreatic trypsin inhibitor (BPTI) is a well-studied protein.
• Understanding protein folding intermediates is crucial for deciphering protein structure-function relationships.
• The role of disulfide bonds in protein stability and folding remains an active area of research.

Purpose of the Study:

• To elucidate the conformational ensemble of [14-38]Abu, a BPTI variant with a single intact disulfide bridge.
• To investigate the structural dynamics and identify key interactions in [14-38]Abu using NMR.
• To model early folding intermediates of BPTI based on the structure of [14-38]Abu.

Main Methods:

• Two-dimensional 1H and 1H-15N NMR spectroscopy were employed to analyze the structure of [14-38]Abu.
• Extensive assignment of NMR spectra, including backbone and side chain atoms and their exchange cross peaks, was performed.
• Analyses included chemical shifts, chemical exchange, hydrogen isotope exchange, and Nuclear Overhauser Effect (NOE) measurements.

Main Results:

• [14-38]Abu exists as an ensemble of interconverting conformations at pH 4.5 and 1°C.
• The 18-24, 29-35 antiparallel beta-sheet remains native-like and intact across all observed conformations.
• Residues outside the beta-sheet undergo local order/disorder transitions, with stable structure localized in a slow-exchange core stabilized by hydrophobic interactions.

Conclusions:

• The stable structure of [14-38]Abu is not dictated by the 14-38 disulfide bond but by a slow-exchange core.
• Tertiary interactions are primarily mediated by hydrophobic contacts involving tyrosine residues (Tyr 21, Tyr 23, Tyr 35).
• [14-38]Abu serves as a valuable model for early folding intermediates, suggesting BPTI folding initiates with turn stabilization and involves hydrophobic interactions.