Analysis of the relationship between enzyme activity and its internal motion using nuclear magnetic resonance: 15N relaxation studies of wild-type and mutant lysozyme

Mine, Shouhei; Tate, Shin Ichi; Ueda, Tadashi; Kainosho, Masatsune; Imoto, Taiji

doi:10.1006/jmbi.1999.2572

Cited by 55 publications

(46 citation statements)

References 43 publications

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“…Second, in studies by Imoto and colleagues (36), deletion of Arg-14 and His-15 at Ն 20 Å from the catalytic center of lysozyme leads to a 1.4-fold increase in chitinase activity (36). Recently, NMR relaxation measurements showed that this mutation altered 15 N-relaxation rates of several residues at or near the active site (37). These studies offer correlative but compelling evidence for the contribution of protein dynamics to enzyme catalysis.…”

Section: Discussionmentioning

confidence: 95%

Changes in protein conformational mobility upon activation of extracellular regulated protein kinase-2 as detected by hydrogen exchange

Hoofnagle

Resing

Goldsmith³

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

106

121

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Changes in protein mobility accompany changes in conformation during the trans-activation of enzymes; however, few studies exist that validate or characterize this behavior. In this study, amide hydrogen/deuterium exchange͞mass spectrometry was used to probe the conformational flexibility of extracellular signal-regulated protein kinase-2 before and after activation by phosphorylation. The exchange data indicated that extracellular regulated protein kinase-2 activation caused altered backbone flexibility in addition to the conformational changes previously established by x-ray crystallography. The changes in flexibility occurred in regions involved in substrate binding and turnover, suggesting their importance in enzyme regulation.T he mitogen-activated protein (MAP) kinase extracellular regulated protein kinase-2 (ERK2) plays a central role in signaling pathways regulating cell growth and differentiation in eukaryotic cells. Phosphorylation at Thr-183 or Tyr-185 by MAP kinase (MAPK) kinase-1͞2 leads to Ͼ1,000-fold enhanced specific kinase activity, resulting in elevated phosphorylation of cellular targets including transcription factors and downstream kinases (1). ERK2 presents an attractive model system for understanding regulated changes involved in enzyme activation, because x-ray structures are available for both inactive, unphosphorylated (ERK) and active diphosphorylated (ppERK) kinases (2, 3).X-ray structures of ERK2 show features conserved in protein kinase catalytic cores (2, 4) (Fig. 1b). These include the Nterminal lobe (␤1-␤5 and helix ␣C) and the larger C-terminal lobe (helices ␣D-␣I and ␤6-␤9), which are responsible for nucleotide and protein substrate binding, respectively, and which surround residues in the catalytic cleft that facilitate phosphoryl transfer. A hinge joins the two lobes and serves as the pivot for domain closure, a conformational change associated with formation of the ternary enzyme-substrate complex (5). The phosphorylation sites in ERK2 are contained in the activation lip, a key site for regulatory phosphorylation and substrate recognition in protein kinases (4, 6).Phosphorylation causes major rearrangements within the activation lip and the adjacent P ϩ 1 protein substrate specificity site (3, 7) (Fig. 1a). These structural rearrangements create a competent substrate binding pocket and properly orient residues necessary for phosphoryl transfer. In addition, phosphorylation induces structural changes in a C-terminal extension (L16), forming a homodimerization interface and stable dimers in active ppERK, but not in inactive ERK (8) (Fig. 1 b and c). Minor structural changes also occur within the MAPK insert (␣1L14 and ␣2L14) where side-chain interactions with the activation lip are disrupted (Fig. 1 b and c).Limited information is available on kinase flexibility and dynamics, factors that are presumably critical to the regulation of enzyme activity. Advances in hydrogen exchange͞mass spectrometry (HX͞MS) enable analysis of solution conformational ensembles for larger proteins ...

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Section: Discussionmentioning

confidence: 95%

Changes in protein conformational mobility upon activation of extracellular regulated protein kinase-2 as detected by hydrogen exchange

Hoofnagle

Resing

Goldsmith³

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

106

121

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“…The construction of single point mutants of hen egg-white lysozyme, the expression and purification of 15 N-labelled protein in the yeast Pichia pastoris, [33,34] and the methylation of reduced lysozyme [35] were performed according to reported methods. CD spectra were recorded as described, [23] and the mean helix content was calculated by using the approach of Rohl and Baldwin.…”

Section: Methodsmentioning

confidence: 99%

Modulation of Compactness and Long‐Range Interactions of Unfolded Lysozyme by Single Point Mutations

et al. 2004

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The non‐native states of the model protein hen lysozyme (native state shown in picture) were investigated by using site‐directed mutagenesis in combination with high‐resolution NMR spectroscopy. The disruptions of the interactions between hydrophobic clusters by single point mutations dramatically alter the overall compactness of the unfolded state: single point mutations can turn a compact unfolded state into an extended state.

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“…Proteins must be flexible enough to allow timely access to the many internal, microscopic and large-scale conformational fluctuations required for function, such as binding and release of a substrate from the interior of an enzyme. [58][59][60] However the propagations of large scale, collective motions that lead to protein unfolding need to be restricted. The connection between the tradeoffs of stability and function then is protein internal dynamics.…”

Section: Proteins In Solution and In The Hydrated Crystalline Statementioning

confidence: 99%

Thermodynamic and dynamic factors involved in the stability of native protein structure in amorphous solids in relation to levels of hydration

Hill

Shalaev

Zografi

2005

Journal of Pharmaceutical Sciences

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Analysis of the relationship between enzyme activity and its internal motion using nuclear magnetic resonance: 15N relaxation studies of wild-type and mutant lysozyme

Cited by 55 publications

References 43 publications

Changes in protein conformational mobility upon activation of extracellular regulated protein kinase-2 as detected by hydrogen exchange

Changes in protein conformational mobility upon activation of extracellular regulated protein kinase-2 as detected by hydrogen exchange

Modulation of Compactness and Long‐Range Interactions of Unfolded Lysozyme by Single Point Mutations

Thermodynamic and dynamic factors involved in the stability of native protein structure in amorphous solids in relation to levels of hydration

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