The dynamics and function of proteins are intimately connected to their three-dimensional structure, which is characterized by fluctuations in atomic positions and conformational transitions covering a broad range of time scales (from sub-picoseconds to seconds) and amplitudes of motion (from sub-angstroms to tens of angstroms). Protein structural changes in solution have so far been characterized mainly by time-resolved optical spectroscopic methods, which give signals that are only indirectly related to three-dimensional structures. For protein crystals, a combination of high time resolution and structural sensitivity became available with the advent of time-resolved Laue crystallography1–3
, but its applicability has been limited to a few model systems because of the stringent prerequisites such as the need for highly ordered and radiation-resistant single crystals. More notably, crystal packing constraints might hinder biologically relevant motions. To obtain information about protein motions in a more natural environment, nuclear magnetic resonance (NMR) and X-ray scattering methods have been used as direct structural probes of protein structure in solution4–6
, but these methods also have limitations. Small-angle X-ray scattering probes the overall size and shape of the protein whereas wide-angle X-ray scattering (WAXS) gives more detailed information such as the fold of helices and sheets7
; however, the time resolution has so far been limited to 160 µs at best8,9
. NMR is a powerful technique for structure determination in solution, but it works best for small proteins, needs properly labeled samples10
, and the time resolution of protein NMR is inherently limited to milliseconds.
Here we demonstrate that time-resolved wide-angle X-ray scattering (TR-WAXS) using synchrotron radiation can be used to accurately probe structural changes of proteins in solution with nanosecond time resolution. TR-WAXS combines the high time resolution already proven to be important for studies on biological samples11,12
with the high structural sensitivity demonstrated for WAXS studies13,14
. TR-WAXS is complementary to time-resolved optical spectroscopy as it allows for tracking of tertiary and quaternary structural changes of a protein with global sensitivity; it is sensitive to changes in the position of all the atoms in the protein rather than to modifications around a given spectroscopic marker. TR-WAXS is applicable to many biologically relevant systems in solution, thus allowing a wide range of experimental parameters such as pH or salt and protein concentrations to be varied.
We report results of TR-WAXS experiments performed mainly on human hemoglobin (Hb), a tetrameric protein made of two identical αβ dimers that is known to adopt at least two different quaternary structures in solution: a ‘relaxed’ (R) structure stabilized by the presence of ligands like CO and O2
, and a ‘tense’ (T) structure that is stable when the protein is unligated15,16
. The ligated-to-unligated transition in Hb involves both conformational changes within the subunits (tertiary structure transition) and changes in the relative disposition of the subunits (quaternary structure transition). The R-T transition has been studied over the last decades and is often used as a paradigm of cooperativity in molecular biology. Our data explain the pathway followed by Hb molecules while switching from the ligated to the unligated state. We obtained information on both the kinetics of the R-T transition and the sequence of structural changes taking place during the transition. In addition, we report preliminary experiments on sperm whale myoglobin (Mb) and horse heart cytochrome c
(Cyt-c); the Mb data show the sensitivity of TR-WAXS to local tertiary conformational changes, and the Cyt-c data demonstrate the applicability of the technique to analyze protein folding.