Micro-Second X-Ray Single Molecule Dynamics of Allosteric Twisting Motions in Hemoglobin

Abstract

Allosteric structural changes underlie the biological functions of many proteins. The best studied is the tense (T) to relaxed (R) state transition in hemoglobin (Hb), which allows for efficient oxygen transport. These two end structures have been solved by crystallography, but little is known about the transition pathway and motions between them. Here we use Diffracted X-ray Tracking (DXT) to monitor single molecule allosteric dynamics in Hb. DXT allows us to monitor the protein motions at high time resolution by tracking diffracted spots from a gold-nanocrystal attached to the protein. X-ray from SPring-8 BL40XU with pink beam was used for the DXT measurements. Data from two rotational directions of the protein molecule, those concerning tilting angle θ and twisting angle χ, are detected. Hb trout III extracted from rainbow trout was used as the sample, which exhibits a remarkable Root effect. It is expected that at pH 8 Hb trout III would exist as an ensemble of the R states with a high oxygen affinity, while at pH 6 it would exist as the T state with a low affinity for oxygen, which is one-thousandth of that of the R state. These unique properties allow us to select conformational populations of Hb by simply adjusting the pH values of the solvents used. Basic time resolution of the system was 100μs/frame. However, we have confirmed that we can observe much faster motions in a solvent containing 30% glycerol at low temperature, where molecular motions of Hb are markedly decelerated. As a result of the DXT observations, we succeeded to clearly detect motions in different twisting direction at a specific moving speed (5-10mard/ms).