Conformational Dependence of Hemoglobin Reactivity under High Viscosity Conditions:  The Role of Solvent Slaved Dynamics

Abstract

The concept of protein dynamic states is introduced. This concept is based on (i) protein dynamics being organized hierarchically with respect to solvent slaving and (ii) which tier of dynamics is operative over the time window of a given measurement. The protein dynamic state concept is used to analyze the kinetic phases derived from the recombination of carbon monoxide to sol-gel-encapsulated human adult hemoglobin (HbA) and select recombinant mutants. The temperature-dependent measurements are made under very high viscosity conditions obtained by bathing the samples in an excess of glycerol. The results are consistent with a given tier of solvent slaved dynamics becoming operative at a time delay (with respect to the onset of the measurement) that is primarily solvent- and temperature-dependent. However, the functional consequences of the dynamics are protein- and conformation-specific. The kinetic traces from both equilibrium populations and trapped allosteric intermediates show a consistent progression that exposes the role of both conformation and hydration in the control of reactivity. Iron-zinc symmetric hybrid forms of HbA are used to show the dramatic difference between the kinetic patterns for T state alpha and beta subunits. The overall results support a model for allostery in HbA in which the ligand-binding-induced transition from the deoxy T state to the high -affinity R state proceeds through a progression of T state intermediates.

Structural and Functional Studies Indicating Altered Redox Properties of Hemoglobin E

“…CO recombination traces from encapsulated R and T state COHb derivatives were generated as described previously (63,65,66). Photodissociation of the CO from the heme is triggered using a 7-ns pulse at 532 nm from a Nd:YAG laser.…”

Immobilization of Proteins in Silica Gel: Biochemical and Biophysical Properties

“…When T state Hb is encapsulated in the absence of allosteric effectors (T -) geminate recombination is detected followed by a biphasic bimolecular rebinding, with characteristics of rebinding to R and T + states [58,89,133,134]. This remarkable finding demonstrates that within the quaternary T state, a significant fraction of subunits behaves as R, and was interpreted on the basis of the tertiary two-state (TTS) model [122,135] as arising from a mixed population of t and r subunits.…”

“…By inhibiting ligand escape to the solvent, this promotes migration to internal cavities and increases the sensitivity of the binding progress curves to intramolecular processes [133,134,137,138]. Several cases were reported where the simple gel entrapment is not enough to increase substantially geminate rebinding.…”

Reverse micelles as a tool for probing solvent modulation of protein dynamics: Reverse micelle encapsulated hemoglobin

“…Functionally important protein dynamics appear to be coupled and possibly slaved to the dynamics of the surrounding solvent [1–7] although questions remain as to the validity of the model [8,9], the generality of the model [10] and its applicability when systems are probed on a micro-site specific level using techniques such as NMR [11,12]. Both protein dynamics and solvent dynamics seemingly possess a hierarchical make-up with respect to amplitudes, time scales and temperature dependences when probed in hemeproteins such as myoglobin and hemoglobin [1,2,8,9,13–16]. The challenging biophysical study of the complex interplay between the different categories of protein dynamics and the various types of solvent motions is an essential aspect in exposing how proteins function.…”

“…To this end, systematically tuning the hydration properties of proteins becomes an important tool in dissecting out how both bulk water and hydration shell waters impact categories of protein dynamics. The present study builds on our earlier work in which sol–gel and glassy matrices are used to systematically tune both protein and solvent dynamics [13,15–22]. Whereas those approaches have the added benefit of allowing for a wide range of temperatures, spanning cryogenic to ambient or even higher, they are limited with respect to being able to fully characterize the nature of the waters surrounding the protein, especially with respect to dimensionality.…”