To understand how a protein functions, it is crucial to know the time-ordered sequence of structural
changes associated with its function. To that end, we have developed numerous experimental
techniques for characterizing structural changes in proteins over time scales ranging from
femtoseconds to seconds. This talk will focus primarily on time-resolved X-ray studies performed on
the BioCARS beamline at the Advanced Photon Source, which allowed us to characterize
structural changes in proteins with 150-ps time resolution. We have used this capability to track the
reversible photocycle of photoactive yellow protein following trans-to-cis photoisomerization of its p-
coumaric acid (pCA) chromophore. A picosecond laser pulse photoexcites pCA and triggers
a structural change in the protein, which is probed with a suitably delayed picosecond X-ray pulse.
When the protein is studied in a crystalline state, this “pump-probe” approach recovers time-resolved
diffraction “snapshots” whose corresponding electron density maps can be stitched together into a
real-time movie of the structural changes that ensue . However, the actual signaling state is not
accessible in the crystalline state due to crystal packing constraints. This state is accessible in time-
resolved small- and wide-angle X-ray scattering studies, which probe changes in the size, shape, and
structure of the protein [2,3]. In order to function, a protein first has to fold into its native structure. We
recently developed a pressure-jump apparatus that allows us to probe protein folding pathways via
NMR methods on the millisecond time scale . These studies help provide a framework for
understanding protein folding and function, and for assessing and validating theoretical/computational
approaches in protein biophysics .
 “Picosecond Photobiology: Watching a Signaling Protein Function in Real Time via Time-resolved
Laue Crystallography,” Friedrich Schotte, Hyun Sun Cho, Ville R I Kaila, Hironari Kamikubo,
Naranbaatar Dashdorj, Eric R Henry, Timothy J. Graber, Rob Henning, Michael Wulff, Gerhard
Hummer, Mikio Kataoka, Philip A. Anfinrud, Proc. Natl. Acad. Sci., USA 109, 19256–19261 (2012).
 “Probing Anisotropic Structure Changes in Proteins with Picosecond Time-Resolved Small-Angle
X-ray Scattering,” Hyun Sun Cho, Friedrich Schotte, Naranbaatar Dashdorj, John Kyndt, and Philip A.
Anfinrud, J Phys Chem B, 117, 15825-15832 (2013).
 “Picosecond Photobiology: Watching a Signaling Protein Function in Real Time via Time-Resolved
Small- and Wide-Angle X-ray Scattering,” Hyun Sun Cho, Friedrich Schotte, Naranbaatar Dashdorj,
John Kyndt, Robert Henning and Philip A. Anfinrud, J. Am. Chem. Soc. 138, 8815-23 (2016).
 “Monitoring Hydrogen Exchange During Protein Folding by Fast Pressure Jump NMR
Spectroscopy,” T. Reid Alderson, Cyril Charlier, Dennis A. Torchia, Philip Anfinrud, and Ad Bax, J.
Am. Chem. Soc. 139, 11036-11039 (2017).
 Contradictions in X-ray structures of intermediates in the photocycle of photoactive yellow protein,”
Ville R. I. Kaila, Friedrich Schotte, Hyun Sun Cho, Gerhard Hummer & Philip A. Anfinrud, Nature
Chemistry, 6, 258 (2014).