Conformational preferences in the Ser<sup>133</sup>‐phosphorylated and non‐phosphorylated forms of the kinase inducible transactivation domain of CREB

Radhakrishnan, Ishwar; Pérez-Alvarado, Gabriela C.; Dyson, H. Jane; Wright, Peter E.

doi:10.1016/s0014-5793(98)00680-2

Cited by 142 publications

(196 citation statements)

References 33 publications

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“…The FEL of the single-chain pKID system showed that the helical propensity for the αB region is considerably smaller than that of the αA region, which is in agreement with experimental results (Radhakrishnan et al 1998). In the pKID-KIX coexisting system, FEL illustrated a number of semi-stable bound forms, including the native-like complex conformation.…”

Section: Applications Of Ttp-mcmd To Interactions Between Intrinsicalsupporting

confidence: 90%

Enhanced sampling simulations to construct free-energy landscape of protein–partner substrate interaction

2016

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Molecular dynamics (MD) simulations using allatom and explicit solvent models provide valuable information on the detailed behavior of protein-partner substrate binding at the atomic level. As the power of computational resources increase, MD simulations are being used more widely and easily. However, it is still difficult to investigate the thermodynamic properties of protein-partner substrate binding and protein folding with conventional MD simulations. Enhanced sampling methods have been developed to sample conformations that reflect equilibrium conditions in a more efficient manner than conventional MD simulations, thereby allowing the construction of accurate free-energy landscapes. In this review, we discuss these enhanced sampling methods using a series of case-by-case examples. In particular, we review enhanced sampling methods conforming to trivial trajectory parallelization, virtual-system coupled multicanonical MD, and adaptive lambda square dynamics. These methods have been recently developed based on the existing method of multicanonical MD simulation. Their applications are reviewed with an emphasis on describing their practical implementation. In our concluding remarks we explore extensions of the enhanced sampling methods that may allow for even more efficient sampling.

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Section: Applications Of Ttp-mcmd To Interactions Between Intrinsicalsupporting

confidence: 90%

Enhanced sampling simulations to construct free-energy landscape of protein–partner substrate interaction

2016

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“…NMR signals of residues around Ser-133 are shifted by the Ser-133-phosphorylation ( Figure 3b). As in vitro phosphorylation has been previously shown to phosphorylate KID specifically at 17], we conclude that Ser-133 of KID is correctly phosphorylated by PKA in E. coli. pKID produced using the in vivo phosphorylation method binds KIX with identical affinity as pKID produced via in vitro phosphorylation (data not shown).…”

Section: Nih-pa Author Manuscriptsupporting

confidence: 73%

“…1 H-15 N heteronuclear single quantum coherence (HSQC) spectra [16] were measured for all samples. The cross peaks of [ 15 N]-KID were identified based on our previous assignments [17].…”

Section: Preparation Of Phosphorylated Kidmentioning

confidence: 99%

Overexpression of post-translationally modified peptides in Escherichia coli by co-expression with modifying enzymes

Sugase¹,

Landes²,

Wright³

et al. 2008

Protein Expression and Purification

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SynopsisPost-translational modification plays crucial roles in signal transduction in eukaryotic cells. To elucidate the biological function of a protein with a specific post-translational modification, it is necessary to isolate the modified protein. However, it is difficult to incorporate a modified amino acid into a specific position of a protein, in particular, in a large-scale preparation. In order to prepare post-translationally modified proteins in Escherichia coli (E. coli), we have constructed coexpression vectors that contain protein and corresponding enzyme genes. The protein and enzyme are co-expressed in the same E. coli cells and the protein is post-translationally modified in vivo. By using this system, the transcriptional activator cyclic-AMP-response-element-binding protein (CREB) was phosphorylated at Ser-133 and the hypoxia-inducible factor-1α (HIF-1α) was hydroxylated at Asn-803 in E. coli. Although the constructs of the proteins we used are very flexible and susceptible to degradation by proteases in E. coli when they are expressed alone, the B1 domain of streptococcal protein G (GB1) fused to the N-terminus of the proteins increased the yields dramatically. Site-specific phosphorylation of CREB and hydroxylation of HIF-1α were confirmed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and NMR. Our GB1-fusion co-expression system can be used in the same way as conventional protein expression in E. coli, making it a flexible and economical method to produce a large amount of a post-translationally modified protein. Keywordsphosphorylation; hydroxylation; co-expression; Escherichia coli; B1 domain of protein G (GB1); CREB; HIF-1α INTRODUCTIONPost-translational modification plays a crucial role in signal transduction in eukaryotic cells by introducing diversity and versatility into proteins translated from genomic DNA sequences [1]. Post-translational modification extends the range of function of the proteins by attaching other biochemical functional groups such as phosphate, hydroxyl group, acetate, lipids, or carbohydrates, causing changes in intrinsic activities of the proteins such as activation, inactivation or change in other properties such as conformational change. Despite the *To whom correspondence should be addressed (email yamout@scripps.edu). † Current address: Suntory Institute for Bioorganic Research, 1-1-1 Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618-8503, Japan.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. importance of post-translational modifications in biology, the production of large quantities of mod...

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“…An R domain composed predominantly of random coil has an analogy in the kinase-inducible domain (KID) of the cAMP-responsive element-binding protein (CREB). Several structural parameters indicate that KID is unstructured (48)(49)(50)(51). Like R8, serine phosphorylation of KID does not increase structure (48,51).…”

Section: Discussionmentioning

confidence: 96%

A functional R domain from cystic fibrosis transmembrane conductance regulator is predominantly unstructured in solution

Ostedgaard

Baldursson

Vermeer

et al. 2000

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

112

114

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Phosphorylation of the regulatory (R) domain initiates cystic fibrosis transmembrane conductance regulator (CFTR) Cl ؊ channel activity. To discover how the function of this domain is determined by its structure, we produced an R domain protein (R8) that spanned residues 708 -831 of CFTR. Phosphorylated, but not unphosphorylated, R8 stimulated activity of CFTR channels lacking this domain, indicating that R8 is functional. Unexpectedly, this functional R8 was predominantly random coil, as revealed by CD and limited proteolysis. The CD spectra of both phosphorylated and nonphosphorylated R8 were similar in aqueous buffer. The folding agent trimethylamine N-oxide induced only a small increase in the helical content of nonphosphorylated R8 and even less change in the helical content of phosphorylated R8. These data, indicating that the R domain is predominantly random coil, may explain the seemingly complex way in which phosphorylation regulates CFTR channel activity.

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Conformational preferences in the Ser¹³³‐phosphorylated and non‐phosphorylated forms of the kinase inducible transactivation domain of CREB

Cited by 142 publications

References 33 publications

Enhanced sampling simulations to construct free-energy landscape of protein–partner substrate interaction

Enhanced sampling simulations to construct free-energy landscape of protein–partner substrate interaction

Overexpression of post-translationally modified peptides in Escherichia coli by co-expression with modifying enzymes

A functional R domain from cystic fibrosis transmembrane conductance regulator is predominantly unstructured in solution

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Conformational preferences in the Ser133‐phosphorylated and non‐phosphorylated forms of the kinase inducible transactivation domain of CREB

Cited by 142 publications

References 33 publications

Enhanced sampling simulations to construct free-energy landscape of protein–partner substrate interaction

Enhanced sampling simulations to construct free-energy landscape of protein–partner substrate interaction

Overexpression of post-translationally modified peptides in Escherichia coli by co-expression with modifying enzymes

A functional R domain from cystic fibrosis transmembrane conductance regulator is predominantly unstructured in solution

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Conformational preferences in the Ser¹³³‐phosphorylated and non‐phosphorylated forms of the kinase inducible transactivation domain of CREB