TY - JOUR
T1 - How internal cavities destabilize a protein
AU - Xue, Mengjun
AU - Wakamoto, Takuro
AU - Kejlberg, Camilla
AU - Yoshimura, Yuichi
AU - Nielsen, Tania Aaquist
AU - Risør, Michael Wulff
AU - Sanggaard, Kristian Wejse
AU - Kitahara, Ryo
AU - Mulder, Frans A.A.
PY - 2019
Y1 - 2019
N2 - Although many proteins possess a distinct folded structure lying at a minimum in a funneled free energy landscape, thermal energy causes any protein to continuously access lowly populated excited states. The existence of excited states is an integral part of biological function. Although transitions into the excited states may lead to protein misfolding and aggregation, little structural information is currently available for them. Here, we show how NMR spectroscopy, coupled with pressure perturbation, brings these elusive species to light. As pressure acts to favor states with lower partial molar volume, NMR follows the ensuing change in the equilibrium spectroscopically, with residue-specific resolution. For T4 lysozyme L99A, relaxation dispersion NMR was used to follow the increase in population of a previously identified "invisible" folded state with pressure, as this is driven by the reduction in cavity volume by the flipping-in of a surface aromatic group. Furthermore, multiple partly disordered excited states were detected at equilibrium using pressure-dependent H/D exchange NMR spectroscopy. Here, unfolding reduced partial molar volume by the removal of empty internal cavities and packing imperfections through subglobal and global unfolding. A close correspondence was found for the distinct pressure sensitivities of various parts of the protein and the amount of internal cavity volume that was lost in each unfolding event. The free energies and populations of excited states allowed us to determine the energetic penalty of empty internal protein cavities to be 36 cal⋅Å-3.
AB - Although many proteins possess a distinct folded structure lying at a minimum in a funneled free energy landscape, thermal energy causes any protein to continuously access lowly populated excited states. The existence of excited states is an integral part of biological function. Although transitions into the excited states may lead to protein misfolding and aggregation, little structural information is currently available for them. Here, we show how NMR spectroscopy, coupled with pressure perturbation, brings these elusive species to light. As pressure acts to favor states with lower partial molar volume, NMR follows the ensuing change in the equilibrium spectroscopically, with residue-specific resolution. For T4 lysozyme L99A, relaxation dispersion NMR was used to follow the increase in population of a previously identified "invisible" folded state with pressure, as this is driven by the reduction in cavity volume by the flipping-in of a surface aromatic group. Furthermore, multiple partly disordered excited states were detected at equilibrium using pressure-dependent H/D exchange NMR spectroscopy. Here, unfolding reduced partial molar volume by the removal of empty internal cavities and packing imperfections through subglobal and global unfolding. A close correspondence was found for the distinct pressure sensitivities of various parts of the protein and the amount of internal cavity volume that was lost in each unfolding event. The free energies and populations of excited states allowed us to determine the energetic penalty of empty internal protein cavities to be 36 cal⋅Å-3.
KW - high-pressure NMR
KW - protein folding and cooperativity
KW - protein stability
KW - unfolded state
UR - http://www.scopus.com/inward/record.url?scp=85073311113&partnerID=8YFLogxK
U2 - 10.1073/pnas.1911181116
DO - 10.1073/pnas.1911181116
M3 - Journal article
C2 - 31570587
AN - SCOPUS:85073311113
SN - 0027-8424
VL - 116
SP - 21031
EP - 21036
JO - Proceedings of the National Academy of Sciences (PNAS)
JF - Proceedings of the National Academy of Sciences (PNAS)
IS - 42
ER -