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今日广西快乐十分开奖:Hydrogen exchange reveals Hsp104 architecture, structural dynamics, and energetics in physiological solution
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The cryo-EM revolution now provides snapshots of the structure of large protein molecular machines, but the fundamental mechanisms and principles that determine how they work have so far been elusive. What is needed is a technology that can investigate their architecture, structural dynamics, and energetics when they are active in physiological solution. This paper demonstrates the use of hydrogen exchange mass spectrometry methods to resolve all of the functionally active and inactive elements of Hsp104, characterize their interactions and dynamics, and uniquely their energetics. The results reveal in broad structural and energy-based terms how Hsp104 works. The methods used, the results obtained, and the energetic considerations that they suggest will be widely applicable to other ATPase-dependent protein machines.
Hsp104 is a large AAA+ molecular machine that can rescue proteins trapped in amorphous aggregates and stable amyloids by drawing substrate protein into its central pore. Recent cryo-EM studies image Hsp104 at high resolution. We used hydrogen exchange mass spectrometry analysis (HX MS) to resolve and characterize all of the functionally active and inactive elements of Hsp104, many not accessible to cryo-EM. At a global level, HX MS confirms the one noncanonical interprotomer interface in the Hsp104 hexamer as a marker for the spiraled conformation revealed by cryo-EM and measures its fast conformational cycling under ATP hydrolysis. Other findings enable reinterpretation of the apparent variability of the regulatory middle domain. With respect to detailed mechanism, HX MS determines the response of each Hsp104 structural element to the different bound adenosine nucleotides (ADP, ATP, AMPPNP, and ATPγS). They are distinguished most sensitively by the two Walker A nucleotide-binding segments. Binding of the ATP analog, ATPγS, tightly restructures the Walker A segments and drives the global open-to-closed/extended transition. The global transition carries part of the ATP/ATPγS-binding energy to the somewhat distant central pore. The pore constricts and the tyrosine and other pore-related loops become more tightly structured, which seems to reflect the energy-requiring directional pull that translocates the substrate protein. ATP hydrolysis to ADP allows Hsp104 to relax back to its lowest energy open state ready to restart the cycle.
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Author contributions: X.Y., J.L., L.M., J.S., and S.W.E. designed research; X.Y. and L.M. performed research; J.L. contributed new reagents/analytic tools; X.Y., L.M., J.S., and S.W.E. analyzed data; and X.Y., J.L., L.M., J.S., and S.W.E. wrote the paper.
Reviewers: L.M.G., University of Massachusetts Amherst; and S.M., University of California, Berkeley.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1816184116/-/DCSupplemental.
Published under the PNAS license.