Additional file 1: of Comparison of glottic views and intubation times in the supine and 25 degree back-up positions

Data Collection Form. (DOCX 189 kb

Direct Observation of Insulin Association Dynamics with Time-Resolved X‑ray Scattering

Biological functions frequently require protein–protein interactions that involve secondary and tertiary structural perturbation. Here we study protein–protein dissociation and reassociation dynamics in insulin, a model system for protein oligomerization. Insulin dimer dissociation into monomers was induced by a nanosecond temperature-jump (T-jump) of ∼8 °C in aqueous solution, and the resulting protein and solvent dynamics were tracked by time-resolved X-ray solution scattering (TRXSS) on time scales of 10 ns to 100 ms. The protein scattering signals revealed the formation of five distinguishable transient species during the association process that deviate from simple two-state kinetics. Our results show that the combination of T-jump pump coupled to TRXSS probe allows for direct tracking of structural dynamics in nonphotoactive proteins

Probing Cytochrome <i>c</i> Folding Transitions upon Phototriggered Environmental Perturbations Using Time-Resolved X‑ray Scattering

Direct tracking of protein structural dynamics during folding–unfolding processes is important for understanding the roles of hierarchic structural factors in the formation of functional proteins. Using cytochrome <i>c</i> (cyt <i>c</i>) as a platform, we investigated its structural dynamics during folding processes triggered by local environmental changes (i.e., pH or heme iron center oxidation/spin/ligation states) with time-resolved X-ray solution scattering measurements. Starting from partially unfolded cyt <i>c</i>, a sudden pH drop initiated by light excitation of a photoacid caused a structural contraction in microseconds, followed by active site restructuring and unfolding in milliseconds. In contrast, the reduction of iron in the heme via photoinduced electron transfer did not affect conformational stability at short timescales (<1 ms), despite active site coordination geometry changes. These results demonstrate how different environmental perturbations can change the nature of interaction between the active site and protein conformation, even within the same metalloprotein, which will subsequently affect the folding structural dynamics

Direct Observation of Cooperative Protein Structural Dynamics of Homodimeric Hemoglobin from 100 ps to 10 ms with Pump–Probe X-ray Solution Scattering

Proteins serve as molecular machines in performing their biological functions, but the detailed structural transitions are difficult to observe in their native aqueous environments in real time. For example, despite extensive studies, the solution-phase structures of the intermediates along the allosteric pathways for the transitions between the relaxed (R) and tense (T) forms have been elusive. In this work, we employed picosecond X-ray solution scattering and novel structural analysis to track the details of the structural dynamics of wild-type homodimeric hemoglobin (HbI) from the clam Scapharca inaequivalvis and its F97Y mutant over a wide time range from 100 ps to 56.2 ms. From kinetic analysis of the measured time-resolved X-ray solution scattering data, we identified three structurally distinct intermediates (I₁, I₂, and I₃) and their kinetic pathways common for both the wild type and the mutant. The data revealed that the singly liganded and unliganded forms of each intermediate share the same structure, providing direct evidence that the ligand photolysis of only a single subunit induces the same structural change as the complete photolysis of both subunits does. In addition, by applying novel structural analysis to the scattering data, we elucidated the detailed structural changes in the protein, including changes in the heme–heme distance, the quaternary rotation angle of subunits, and interfacial water gain/loss. The earliest, R-like I₁ intermediate is generated within 100 ps and transforms to the R-like I₂ intermediate with a time constant of 3.2 ± 0.2 ns. Subsequently, the late, T-like I₃ intermediate is formed via subunit rotation, a decrease in the heme–heme distance, and substantial gain of interfacial water and exhibits ligation-dependent formation kinetics with time constants of 730 ± 120 ns for the fully photolyzed form and 5.6 ± 0.8 μs for the partially photolyzed form. For the mutant, the overall kinetics are accelerated, and the formation of the T-like I₃ intermediate involves interfacial water loss (instead of water entry) and lacks the contraction of the heme–heme distance, thus underscoring the dramatic effect of the F97Y mutation. The ability to keep track of the detailed movements of the protein in aqueous solution in real time provides new insights into the protein structural dynamics

X-ray scattering data for: Sequential conformational transitions and alpha-helical supercoiling regulate a sensor histidine kinase

X-ray solution scattering data collected at BioCARS at APS and cSAXS at SLS respectively. The datasets have been normalized but not scaled to eachother. The solvent heating contribution has not been subtracted and the cSAXS data have not been convoluted with the BioCARS wavelength distribution. The data are provided as comma separated values with the q-vector to the far left and laser pump X-ray probe time-delays as ns (BioCARS) or s (cSAXS) in the first row (not applicable to heat data files)