Modulation of Functionally Significant Conformational Equilibria in Adenylate Kinase by High Concentrations of Trimethylamine Oxide Attributed to Volume Exclusion

Nagarajan, Sureshbabu; Amir, Dan; Grupi, Asaf; Goldenberg, David P.; Minton, Allen P.; Haas, Elisha

doi:10.1016/j.bpj.2011.03.065

Cited by 29 publications

(41 citation statements)

References 54 publications

(62 reference statements)

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“…For the opening−closing event, it has been shown with time-resolved FRET experiments that TMAO addition reduces the distance between the side chains of residue 142 in the ATPlid and residue 203 in the CORE subdomain. 51 This nicely illustrates that closed conformations are favored in presence of TMAO. A 1 H− 15 N HSQC spectrum of AK eco in presence 0.35 M TMAO is of high quality (Supporting Information Figure 1C) and the overall chemical perturbations referenced to AK eco under regular buffer conditions are relatively small (Supporting Information Figure 1D).…”

Section: ■ Results and Discussionmentioning

confidence: 83%

“…12,29 . 52 Since FRET experiments have shown that TMAO affects the open/closed equilibrium by stabilizing closed conformations, 51 we conclude that both the hydrogen bond and TMAO perturbations affect the open/closed equilibrium. In all cases, the chemical shifts of apo AK eco appear in-between those of the two perturbed states.…”

Section: ■ Results and Discussionmentioning

confidence: 96%

“…Finally, FRET experiments have shown that TMAO favors closed AK eco states 51. It can also not be ruled out that the microscopic mechanisms of AK eco catalysis are altered in the presence of TMAO.…”

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confidence: 98%

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Modulation of a Pre-existing Conformational Equilibrium Tunes Adenylate Kinase Activity

et al. 2012

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Structural plasticity is often required for distinct microscopic steps during enzymatic reaction cycles. Adenylate kinase from Escherichia coli (AK(eco)) populates two major conformations in solution; the open (inactive) and closed (active) state, and the overall turnover rate is inversely proportional to the lifetime of the active conformation. Therefore, structural plasticity is intimately coupled to enzymatic turnover in AK(eco). Here, we probe the open to closed conformational equilibrium in the absence of bound substrate with NMR spectroscopy and molecular dynamics simulations. The conformational equilibrium in absence of substrate and, in turn, the turnover number can be modulated with mutational- and osmolyte-driven perturbations. Removal of one hydrogen bond between the ATP and AMP binding subdomains results in a population shift toward the open conformation and a resulting increase of k(cat). Addition of the osmolyte TMAO to AK(eco) results in population shift toward the closed conformation and a significant reduction of k(cat). The Michaelis constants (K(M)) scale with the change in k(cat), which follows from the influence of the population of the closed conformation for substrate binding affinity. Hence, k(cat) and K(M) are mutually dependent, and in the case of AK(eco), any perturbation that modulates k(cat) is mirrored with a proportional response in K(M). Thus, our results demonstrate that the equilibrium constant of a pre-existing conformational equilibrium directly affects enzymatic catalysis. From an evolutionary perspective, our findings suggest that, for AK(eco), there exists ample flexibility to obtain a specificity constant (k(cat)/K(M)) that commensurate with the exerted cellular selective pressure.

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Section: ■ Results and Discussionmentioning

confidence: 83%

Section: ■ Results and Discussionmentioning

confidence: 96%

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Modulation of a Pre-existing Conformational Equilibrium Tunes Adenylate Kinase Activity

et al. 2012

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“…This is possible for proteins where a significant difference in SAS area exists between the structural states involved. Urea has opposite effects on these dynamics to those of osmolytes like TMAO, which favor the closed and more compact state of Adk at the expense of the open, more expanded state (10,38). Previous studies involving the FIGURE 13 (A) Illustrative projection amplitudes (X) for three amino acid residues (57, red; 124, blue; and 176, green).…”

Section: Discussionmentioning

confidence: 99%

Urea-Dependent Adenylate Kinase Activation following Redistribution of Structural States

Rogne

Wolf‐Watz

2016

Biophysical Journal

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Proteins are often functionally dependent on conformational changes that allow them to sample structural states that are sparsely populated in the absence of a substrate or binding partner. The distribution of such structural microstates is governed by their relative stability, and the kinetics of their interconversion is governed by the magnitude of associated activation barriers. Here, we have explored the interplay among structure, stability, and function of a selected enzyme, adenylate kinase (Adk), by monitoring changes in its enzymatic activity in response to additions of urea. For this purpose we used a 31 P NMR assay that was found useful for heterogeneous sample compositions such as presence of urea. It was found that Adk is activated at low urea concentrations whereas higher urea concentrations unfolds and thereby deactivates the enzyme. From a quantitative analysis of chemical shifts, it was found that urea redistributes preexisting structural microstates, stabilizing a substrate-bound open state at the expense of a substrate-bound closed state. Adk is rate-limited by slow opening of substrate binding domains and the urea-dependent redistribution of structural states is consistent with a model where the increased activity results from an increased rate-constant for domain opening. In addition, we also detected a strong correlation between the catalytic free energy and free energy of substrate (ATP) binding, which is also consistent with the catalytic model for Adk. From a general perspective, it appears that urea can be used to modulate conformational equilibria of folded proteins toward more expanded states for cases where a sizeable difference in solvent-accessible surface area exists between the states involved. This effect complements the action of osmolytes, such as trimethylamine N-oxide, that favor more compact protein states.

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“…Adk undergoes a large conformational change in response to ligand binding that shuttles the protein into a closed and active conformation where phosphoryl transfer occurs (Rhoads & Lowenstein, 1968). Because of its favorable properties -specifically, its tendency to yield excellent NMR spectra (Ådén & Wolf-Watz, 2007;Schrank et al 2009), catalysis of a reversible reaction, and large conformational changes (Müller et al 1996;Müller & Schulz, 1992) -Adk has emerged as one of the principal model systems for analyzing the linkage between dynamics and enzymatic turnover (Arora & Brooks, 2007;Hanson et al 2007;Nagarajan et al 2011;Schrank et al 2009;Shapiro et al 2009Shapiro et al , 2000Shapiro & Meirovitch, 2006). In an extensive multi-approach effort, the Kern laboratory has shown that substrate-free Adk from the hyperthermophilic bacterium Aquifex aeolicus samples 'closed like' structures (Henzler-Wildman et al 2007).…”

Section: Adkmentioning

confidence: 99%

Protein dynamics and function from solution state NMR spectroscopy

Kovermann

Rogne

Wolf‐Watz

2016

Quart. Rev. Biophys.

143

122

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Abstract. It is well-established that dynamics are central to protein function; their importance is implicitly acknowledged in the principles of the Monod, Wyman and Changeux model of binding cooperativity, which was originally proposed in 1965. Nowadays the concept of protein dynamics is formulated in terms of the energy landscape theory, which can be used to understand protein folding and conformational changes in proteins. Because protein dynamics are so important, a key to understanding protein function at the molecular level is to design experiments that allow their quantitative analysis. Nuclear magnetic resonance (NMR) spectroscopy is uniquely suited for this purpose because major advances in theory, hardware, and experimental methods have made it possible to characterize protein dynamics at an unprecedented level of detail. Unique features of NMR include the ability to quantify dynamics (i) under equilibrium conditions without external perturbations, (ii) using many probes simultaneously, and (iii) over large time intervals. Here we review NMR techniques for quantifying protein dynamics on fast (ps-ns), slow (μs-ms), and very slow (s-min) time scales. These techniques are discussed with reference to some major discoveries in protein science that have been made possible by NMR spectroscopy.

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Modulation of Functionally Significant Conformational Equilibria in Adenylate Kinase by High Concentrations of Trimethylamine Oxide Attributed to Volume Exclusion

Cited by 29 publications

References 54 publications

Modulation of a Pre-existing Conformational Equilibrium Tunes Adenylate Kinase Activity

Modulation of a Pre-existing Conformational Equilibrium Tunes Adenylate Kinase Activity

Urea-Dependent Adenylate Kinase Activation following Redistribution of Structural States

Protein dynamics and function from solution state NMR spectroscopy

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