Allosteric action in real time: Time-resolved crystallographic studies of a cooperative dimeric hemoglobin

Abstract

Protein allostery provides mechanisms for regulation of biological function at the molecular level. We present here an investigation of global, ligand-induced allosteric transition in a protein by time-resolved x-ray diffraction. The study provides a view of structural changes in single crystals of Scapharca dimeric hemoglobin as they proceed in real time, from 5 ns to 80 s after ligand photodissociation. A tertiary intermediate structure forms rapidly (<5 ns) as the protein responds to the presence of an unliganded heme within each R-state protein subunit, with key structural changes observed in the heme groups, neighboring residues, and interface water molecules. This intermediate lays a foundation for the concerted tertiary and quaternary structural changes that occur on a microsecond time scale and are associated with the transition to a low-affinity T-state structure. Reversal of these changes shows a considerable lag as a T-like structure persists well after ligand rebinding, suggesting a slow T-to-R transition.allosteric protein transitions ͉ intersubunit communication ͉ kinetics A fundamental goal in biology is to understand how organisms respond to environmental signals. Central to this process are allosteric proteins that undergo structural transitions between alternate states in response to stimuli such as ligand binding. Although static structures of alternate states are available for a number of allosteric proteins, information about the kinetic pathway between such functionally important states is limited. Time-resolved crystallographic analysis (1-9) provides the unique opportunity to obtain direct, time-dependent structural information at high resolution on the entire protein molecule as it undergoes structural change.Much of our understanding of allosteric protein function has come from investigations into mammalian hemoglobins. Evidence of substantial structural changes accompanying ligand binding dates back to the 1930s with the observation that unliganded, high-salt, hemoglobin crystals exposed to oxygen shatter (10), foreshadowing the discovery of large quaternary changes that are linked to oxygen binding (11). Unliganded crystals of human hemoglobin grown under certain low-salt conditions can maintain their integrity upon oxygen binding; however, oxygen binding in this case is noncooperative (12). Although the ability to follow allosteric transitions in the crystalline state is limited when such large quaternary structural changes accompany cooperative ligand binding, spectroscopic investigations of hemoglobin solutions have revealed some aspects of the kinetic pathway of allosteric structural transitions (13-15). Results to date suggest a multistep pathway, with key tertiary structural transitions taking place within a few microseconds and quaternary rearrangements occurring in the range of tens of microseconds. In addition to providing structural information on high-affinity R and low-affinity T quaternary forms at the atomic level (16-18), crystallographic experiments have also succeeded...

An Optical Signal Correlated with the Allosteric Transition in Scapharca inaequivalvis HbI

“…Among these changes, the heme macrocycle resonance at 756cm −1 and the iron-proximal histidine stretching mode at 203cm −1 may be correlated with the R to T transition of Phe 97 and thus may be related to the rts signal observed here. The rts time course is slightly faster than suggested by time-resolved crystallography (13), which probably reflects real differences between crystalline and solution experiments.…”

Ligand Migration and Binding in the Dimeric Hemoglobin ofScapharca inaequivalvis,

“…Here we propose a similar scenario for wild-type HbICO. Time-resolved x-ray crystallography experiments on M37V HbICO yielded a positive electron density in the distal pocket adjacent to the Val-37 (B10) side chain 5 ns after photolysis due to photolyzed CO (85). This position is similar to the photoproduct site B of Mb.…”

Allostery and cooperativity revisited

“…Since, unlike HbA, no structures for intermediates or their analogs are available, they were constructed by combining subunits from the unliganded and liganded dimers; because of the small structural changes between the two species, little relaxation was required to obtain a stable structure. A recent time-resolved X-ray study of the allosteric transition in HbI (Knapp et al 2006) gives some information about the intermediate and it is hoped that a structure for it will be forthcoming (W. Royer Jr., pers. comm.).…”