共查询到20条相似文献,搜索用时 15 毫秒
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Masahiro Kitazawa Yoshihiro Kubo Koichi Nakajo 《The Journal of biological chemistry》2015,290(37):22724-22733
Kv4 is a member of the voltage-gated K+ channel family and forms a complex with various accessory subunits. Dipeptidyl aminopeptidase-like protein (DPP) is one of the auxiliary subunits for the Kv4 channel. Although DPP has been well characterized and is known to increase the current amplitude and accelerate the inactivation and recovery from inactivation of Kv4 current, it remains to be determined how many DPPs bind to one Kv4 channel. To examine whether the expression level of DPP changes the biophysical properties of Kv4, we expressed Kv4.2 and DPP10 in different ratios in Xenopus oocytes and analyzed the currents under two-electrode voltage clamp. The current amplitude and the speed of recovery from inactivation of Kv4.2 changed depending on the co-expression level of DPP10. This raised the possibility that the stoichiometry of the Kv4.2-DPP10 complex is variable and affects the biophysical properties of Kv4.2. We next determined the stoichiometry of DPP10 alone by subunit counting using single-molecule imaging. Approximately 70% of the DPP10 formed dimers in the plasma membrane, and the rest existed as monomers in the absence of Kv4.2. We next determined the stoichiometry of the Kv4.2-DPP10 complex; Kv4.2-mCherry and mEGFP-DPP10 were co-expressed in different ratios and the stoichiometries of Kv4.2-DPP10 complexes were evaluated by the subunit counting method. The stoichiometry of the Kv4.2-DPP10 complex was variable depending on the relative expression level of each subunit, with a preference for 4:2 stoichiometry. This preference may come from the bulky dimeric structure of the extracellular domain of DPP10. 相似文献
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A neuronal Ca2+/calmodulin-dependent protein kinase (CaM kinase-Gr) undergoes autophosphorylation on a serine residue(s) in response to Ca2+ and calmodulin. Phosphate incorporation leads to the formation of a Ca(2+)-independent (autonomous) activity state, as well as potentiation of the Ca2+/calmodulin-dependent response. The autonomous enzyme activity of the phosphorylated enzyme approximately equals the Ca2+/calmodulin-stimulated activity of the unphosphorylated enzyme, but displays diminished affinity toward ATP and the synthetic substrate, syntide-2. The Km(app) for ATP and syntide-2 increased 4.3- and 1.7-fold, respectively. Further activation of the autonomous enzyme by Ca2+/calmodulin yields a marked increase in the affinity for ATP and peptide substrate such that the Km(app) for ATP and syntide-2 decreased by 14- and 8-fold, respectively. Both autophosphorylation and the addition of Ca2+/calmodulin are required to produce the maximum level of enzyme activation and to increase substrate affinity. Unlike Ca2+/calmodulin-dependent protein kinase type II that is dephosphorylated by the Mg(2+)-independent phosphoprotein phosphatases 1 and 2A, CaM kinase-Gr is dephosphorylated by a Mg(2+)-dependent phosphoprotein phosphatase that may be related to the type 2C enzyme. Dephosphorylation of CaM kinase-Gr reverses the effects of autophosphorylation on enzyme activity. A comparison between the autophosphorylation and dephosphorylation reactions of CaM kinase-Gr and Ca2+/calmodulin-dependent protein kinase type II provides useful insights into the operation of Ca(2+)-sensitive molecular switches. 相似文献
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Assembly of the eukaryotic ribosome requires a large number of trans-acting proteins and small nucleolar RNAs that transiently associate with the precursor rRNA to facilitate its modification, processing and binding with ribosomal proteins. UTPB is a large evolutionarily conserved complex in the 90S small subunit processome that mediates early processing of 18S rRNA. UTPB consists of six proteins Utp1/Pwp1, Utp6, Utp12/Dip2, Utp13, Utp18 and Utp21 and has abundant WD domains. Here, we determined the crystal structure of the tandem WD domain of yeast Utp21 at 2.1 Å resolution, revealing two open-clamshell-shaped β-propellers. The bottom faces of both WD domains harbor several conserved patches that potentially function as molecular binding sites. We show that residues 100–190 of Utp18 bind to the tandem WD domain of Utp21. Structural mapping of previous crosslinking data shows that the WD domains of Utp18 and Utp1 are organized on two opposite sides of the Utp21 WD domains. This study reports the first structure of a UTPB component and provides insight into the structural organization of the UTPB complex. 相似文献
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All subtypes of KCNQ channel subunits (KCNQ1-5) require calmodulin as a co-factor for functional channels. It has been demonstrated that calmodulin plays a critical role in KCNQ channel trafficking as well as calcium-mediated current modulation. However, how calcium-bound calmodulin suppresses the M-current is not well understood. In this study, we investigated the molecular mechanism of KCNQ2 current suppression mediated by calcium-bound calmodulin. We show that calcium induced slow calmodulin dissociation from the KCNQ2 channel subunit. In contrast, in homomeric KCNQ3 channels, calcium facilitated calmodulin binding. We demonstrate that this difference in calmodulin binding was due to the unique cysteine residue in the KCNQ2 subunit at aa 527 in Helix B, which corresponds to an arginine residue in other KCNQ subunits including KCNQ3. In addition, a KCNQ2 channel associated protein AKAP79/150 (79 for human, 150 for rodent orthologs) also preferentially bound calcium-bound calmodulin. Therefore, the KCNQ2 channel complex was able to retain calcium-bound calmodulin either through the AKPA79/150 or KCNQ3 subunit. Functionally, increasing intracellular calcium by ionomycin suppressed currents generated by KCNQ2, KCNQ2(C527R) or heteromeric KCNQ2/KCNQ3 channels to an equivalent extent. This suggests that a change in the binding configuration, rather than dissociation of calmodulin, is responsible for KCNQ current suppression. Furthermore, we demonstrate that KCNQ current suppression was accompanied by reduced KCNQ affinity toward phosphatidylinositol 4,5-bisphosphate (PIP2) when assessed by a voltage-sensitive phosphatase, Ci-VSP. These results suggest that a rise in intracellular calcium induces a change in the configuration of CaM-KCNQ binding, which leads to the reduction of KCNQ affinity for PIP2 and subsequent current suppression. 相似文献
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Expression of a neuronal Ca2+/calmodulin-dependent protein kinase, CaM kinase-Gr, in rat thymus 总被引:2,自引:0,他引:2
M V Frangakis T Chatila E R Wood N Sahyoun 《The Journal of biological chemistry》1991,266(26):17592-17596
The regional and tissue-specific expression of the Ca2+/calmodulin-dependent protein kinase, CaM kinase-Gr, were examined. The Mr 65,000 alpha-polypeptide of CaM kinase-Gr is expressed ubiquitously in different anatomical regions of rat brain, whereas an additional Mr 67,000 beta-polypeptide is observed solely in the cerebellum. The alpha-polypeptide appears in the neonatal rat forebrain and cerebellum, whereas the beta-polypeptide appears by the second postnatal week and may reflect cerebellar granule cell differentiation. Most peripheral tissues do not express either CaM kinase-Gr polypeptide. However, rat thymus and thymocytes derived therefrom express CaM kinase-Gr at levels comparable to those of the central nervous system. The identity of the enzyme in rat thymus was corroborated by immunoblot assays, Northern blots, and direct enzyme purification. Rat spleen and testis also produce CaM kinase-Gr, but at lower levels than either thymus or brain. These observations demonstrate selective regional and developmental expression of CaM kinase-Gr polypeptide in brain, and suggest that it may participate in Ca2+ signalling in cells derived both from the immune system as well as the central nervous system. 相似文献
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Zhubing Shi Shi Jiao Zhen Zhang Miao Ma Zhao Zhang Cuicui Chen Ke Wang Huizhen Wang Wenjia Wang Lei Zhang Yun Zhao Zhaocai Zhou 《Structure (London, England : 1993)》2013,21(3):449-461
Highlights? MO25 forms a stable complex with MST4 to enhance its kinase activity ? Association of MO25 activates MST4 by rotating its αC helix to active conformation ? Kinase-domain-mediated dimerization is required for MST4 trans-autophosphorylation ? MO25-stimulated activation of MST4 promotes apoptosis in HEK293T cells 相似文献
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Heng Zhang Heng Zhang Zeng-Qiang Gao Wen-Jia Wang Guang-Feng Liu Jian-Hua Xu Xiao-Dong Su Yu-Hui Dong 《The Journal of biological chemistry》2013,288(8):5928-5939
The type VI secretion system (T6SS), a multisubunit needle-like apparatus, has recently
been found to play a role in interspecies interactions. The Gram-negative bacteria
harboring T6SS (donor) deliver the effectors into their neighboring cells (recipient) to
kill them. Meanwhile, the cognate immunity proteins were employed to protect the donor
cells against the toxic effectors. Tae4 (type VI
amidase effector 4) and Tai4
(type VI amidase
immunity 4) are newly identified T6SS effector-immunity pairs.
Here, we report the crystal structures of Tae4 from Enterobacter cloacae
and Tae4-Tai4 complexes from both E. cloacae and Salmonella
typhimurium. Tae4 acts as a dl-endopeptidase and displays a typical
N1pC/P60 domain. Unlike Tsi1 (type VI
secretion immunity 1), Tai4 is an
all-helical protein and forms a dimer in solution. The small angle x-ray scattering study
combined with the analytical ultracentrifugation reveal that the Tae4-Tai4 complex is a
compact heterotetramer that consists of a Tai4 dimer and two Tae4 molecules in solution.
Structure-based mutational analysis of the Tae4-Tai4 interface shows that a helix
(α3) of one subunit in dimeric Tai4 plays a major role in binding of Tae4, whereas
a protruding loop (L4) in the other subunit is mainly responsible for inhibiting Tae4
activity. The inhibition process requires collaboration between the Tai4 dimer. These
results reveal a novel and unique inhibition mechanism in effector-immunity pairs and
suggest a new strategy to develop antipathogen drugs. 相似文献
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Julien Schelpe Didier Monté Frédérique Dewitte Titia K. Sixma Prakash Rucktooa 《The Journal of biological chemistry》2016,291(2):630-639
FAT10 conjugation, a post-translational modification analogous to ubiquitination, specifically requires UBA6 and UBE2Z as its activating (E1) and conjugating (E2) enzymes. Interestingly, these enzymes can also function in ubiquitination. We have determined the crystal structure of UBE2Z and report how the different domains of this E2 enzyme are organized. We further combine our structural data with mutational analyses to understand how specificity is achieved in the FAT10 conjugation pathway. We show that specificity toward UBA6 and UBE2Z lies within the C-terminal CYCI tetrapeptide in FAT10. We also demonstrate that this motif slows down transfer rates for FAT10 from UBA6 onto UBE2Z. 相似文献
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《Journal of molecular biology》2019,431(7):1440-1459
Calcium/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional serine/threonine protein kinase that transmits calcium signals in various cellular processes. CaMKII is activated by calcium-bound calmodulin (Ca2+/CaM) through a direct binding mechanism involving a regulatory C-terminal α-helix in CaMKII. The Ca2+/CaM binding triggers transphosphorylation of critical threonine residues proximal to the CaM-binding site leading to the autoactivated state of CaMKII. The demonstration of its critical roles in pathophysiological processes has elevated CaMKII to a key target in the management of numerous diseases. The molecule KN-93 is the most widely used inhibitor for studying the cellular and in vivo functions of CaMKII. It is widely believed that KN-93 binds directly to CaMKII, thus preventing kinase activation by competing with Ca2+/CaM. Herein, we employed surface plasmon resonance, NMR, and isothermal titration calorimetry to characterize this presumed interaction. Our results revealed that KN-93 binds directly to Ca2+/CaM and not to CaMKII. This binding would disrupt the ability of Ca2+/CaM to interact with CaMKII, effectively inhibiting CaMKII activation. Our findings also indicated that KN-93 can specifically compete with a CaMKIIδ-derived peptide for binding to Ca2+/CaM. As indicated by the surface plasmon resonance and isothermal titration calorimetry data, apparently at least two KN-93 molecules can bind to Ca2+/CaM. Our findings provide new insight into how in vitro and in vivo data obtained with KN-93 should be interpreted. They further suggest that other Ca2+/CaM-dependent, non-CaMKII activities should be considered in KN-93–based mechanism-of-action studies and drug discovery efforts. 相似文献
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Sascha A. Walzer Claudia Egerer-Sieber Heinrich Sticht Madhumati Sevvana Katharina Hohl Jens Milbradt Yves A. Muller Manfred Marschall 《The Journal of biological chemistry》2015,290(46):27452-27458
Nuclear replication of cytomegalovirus relies on elaborate mechanisms of nucleocytoplasmic egress of viral particles. Thus, the role of two essential and conserved viral nuclear egress proteins, pUL50 and pUL53, is pivotal. pUL50 and pUL53 heterodimerize and form a core nuclear egress complex (NEC), which is anchored to the inner nuclear membrane and provides a scaffold for the assembly of a multimeric viral-cellular NEC. Here, we report the crystal structure of the pUL50-pUL53 heterodimer (amino acids 1–175 and 50–292, respectively) at 2.44 Å resolution. Both proteins adopt a globular fold with mixed α and β secondary structure elements. pUL53-specific features include a zinc-binding site and a hook-like N-terminal extension, the latter representing a hallmark element of the pUL50-pUL53 interaction. The hook-like extension (amino acids 59–87) embraces pUL50 and contributes 1510 Å2 to the total interface area (1880 Å2). The pUL50 structure overall resembles the recently published NMR structure of the murine cytomegalovirus homolog pM50 but reveals a considerable repositioning of the very C-terminal α-helix of pUL50 upon pUL53 binding. pUL53 shows structural resemblance with the GHKL domain of bacterial sensory histidine kinases. A close examination of the crystal structure indicates partial assembly of pUL50-pUL53 heterodimers to hexameric ring-like structures possibly providing additional scaffolding opportunities for NEC. In combination, the structural information on pUL50-pUL53 considerably improves our understanding of the mechanism of HCMV nuclear egress. It may also accelerate the validation of the NEC as a unique target for developing a novel type of antiviral drug and improved options of broad-spectrum antiherpesviral therapy. 相似文献
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Michael K. Uhl Gustav Oberdorfer Georg Steinkellner Lina Riegler-Berket Daniel Mink Friso van Assema Martin Schürmann Karl Gruber 《PloS one》2015,10(4)
Threonine aldolases catalyze the pyridoxal phosphate (PLP) dependent cleavage of threonine into glycine and acetaldehyde and play a major role in the degradation of this amino acid. In nature, L- as well as D-specific enzymes have been identified, but the exact physiological function of D-threonine aldolases (DTAs) is still largely unknown. Both types of enantio-complementary enzymes have a considerable potential in biocatalysis for the stereospecific synthesis of various β-hydroxy amino acids, which are valuable building blocks for the production of pharmaceuticals. While several structures of L-threonine aldolases (LTAs) have already been determined, no structure of a DTA is available to date. Here, we report on the determination of the crystal structure of the DTA from Alcaligenes xylosoxidans (AxDTA) at 1.5 Å resolution. Our results underline the close relationship of DTAs and alanine racemases and allow the identification of a metal binding site close to the PLP-cofactor in the active site of the enzyme which is consistent with the previous observation that divalent cations are essential for DTA activity. Modeling of AxDTA substrate complexes provides a rationale for this metal dependence and indicates that binding of the β-hydroxy group of the substrate to the metal ion very likely activates this group and facilitates its deprotonation by His193. An equivalent involvement of a metal ion has been implicated in the mechanism of a serine dehydratase, which harbors a metal ion binding site in the vicinity of the PLP cofactor at the same position as in DTA. The structure of AxDTA is completely different to available structures of LTAs. The enantio-complementarity of DTAs and LTAs can be explained by an approximate mirror symmetry of crucial active site residues relative to the PLP-cofactor. 相似文献
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Luminescence enhancement of (Sr1–xMx)2SiO4:Eu2+ phosphors with M (Ca2+/Zn2+) partial substitution for white light‐emitting diodes 下载免费PDF全文
Eu2+‐doped Sr2SiO4 phosphor with Ca2+/Zn2+ substitution, (Sr1–xMx)2SiO4:Eu2+ (M = Ca, Zn), was prepared using a high‐temperature solid‐state reaction method. The structure and luminescence properties of Ca2+/Zn2+ partially substituted Sr2SiO4:Eu2+ phosphors were investigated in detail. With Ca2+ or Zn2+ added to the silicate host, the crystal phase could be transformed between the α‐form and the β‐form of the Sr2SiO4 structure. Under UV excitation at 367 nm, all samples exhibit a broad band emission from 420 to 680 nm due to the 4f65d1 → 4f7 transition of Eu2+ ions. The broad emission band consists of two peaks at 482 and 547 nm, which correspond to Eu2+ ions occupying the ten‐fold oxygen‐coordinated Sr.(I) site and the nine‐fold oxygen‐coordinated Sr.(II) site, respectively. The luminescence properties, including the intensity and lifetime of Sr2SiO4:Eu2+ phosphors, improved remarkably on Ca2+/Zn2+ addition, and promote its application in white light‐emitting diodes. Copyright © 2016 John Wiley & Sons, Ltd. 相似文献
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Vincent Chaptal Michela Ottolia Gabriel Mercado-Besserer Debora A. Nicoll Kenneth D. Philipson Jeff Abramson 《The Journal of biological chemistry》2009,284(22):14688-14692
The mammalian Na+/Ca2+ exchanger, NCX1.1, serves as
the main mechanism for Ca2+ efflux across the sarcolemma following
cardiac contraction. In addition to transporting Ca2+, NCX1.1
activity is also strongly regulated by Ca2+ binding to two
intracellular regulatory domains, CBD1 and CBD2. The structures of both of
these domains have been solved by NMR spectroscopy and x-ray crystallography,
greatly enhancing our understanding of Ca2+ regulation.
Nevertheless, the mechanisms by which Ca2+ regulates the exchanger
remain incompletely understood. The initial NMR study showed that the first
regulatory domain, CBD1, unfolds in the absence of regulatory Ca2+.
It was further demonstrated that a mutation of an acidic residue involved in
Ca2+ binding, E454K, prevents this structural unfolding. A
contradictory result was recently obtained in a second NMR study in which
Ca2+ removal merely triggered local rearrangements of CBD1. To
address this issue, we solved the crystal structure of the E454K-CBD1 mutant
and performed electrophysiological analyses of the full-length exchanger with
mutations at position 454. We show that the lysine substitution replaces the
Ca2+ ion at position 1 of the CBD1 Ca2+ binding site and
participates in a charge compensation mechanism. Electrophysiological analyses
show that mutations of residue Glu-454 have no impact on Ca2+
regulation of NCX1.1. Together, structural and mutational analyses indicate
that only two of the four Ca2+ ions that bind to CBD1 are important
for regulating exchanger activity.Cardiac contraction/relaxation relies upon Ca2+ fluxes across
the plasma membrane (sarcolemma) of cardiomyocytes. Rapid Ca2+
influx (primarily through L-type Ca2+ channels) triggers the
release of additional Ca2+ from the sarcoplasmic reticulum
(SR),4 resulting in
cardiomyocyte contraction. Removal of cytosolic Ca2+ by reuptake
into the SR (through the SR Ca2+-ATPase) and expulsion from the
cell (primarily through the Na+/Ca2+ exchanger, NCX1.1)
results in relaxation (1).
Altered Ca2+ cycling is observed in a number of pathophysiological
situations including ischemia, hypertrophy, and heart failure
(2). Understanding the function
and regulation of NCX1.1 is thus of fundamental importance to understand
cardiac physiology.NCX1.1 utilizes the electrochemical potential of the Na+
gradient to extrude Ca2+ in a ratio of three Na+ ions to
one Ca2+ ion (3). In
addition to transporting both Na+ and Ca2+, NCX1.1 is
also strongly regulated by these two ions. Intracellular Na+ can
induce NCX1.1 to enter an inactivated state, whereas Ca2+ bound to
regulatory sites removes Na+-dependent inactivation and also
activates Na+/Ca2+ exchange
(3). These regulatory sites are
located on a large cytoplasmic loop (∼500 residues located between
transmembrane helices V and VI) containing two calcium binding domains (CBD1
and CBD2), which sense cytosolic Ca2+ levels. We have previously
shown that Ca2+ binding to the primary site in CBD2 is required for
full exchange regulation (4);
CBD1, however, is a site of higher affinity and appears to dominate the
activation of exchange activity by Ca2+.Both CBDs have an immunoglobulin fold formed from two antiparallel β
sheets generating a β sandwich with a differing number of Ca2+
ions coordinated at the tip of the domain
(4,
5). CBD1 binds four
Ca2+ ions, whereas CBD2 binds only two Ca2+ ions. An
initial NMR study revealed a local unfolding of the upper portion of CBD1 upon
release of Ca2+ (6).
In contrast, CBD2 did not display an unfolding response upon Ca2+
removal. A comparative analysis between CBDs revealed a difference in charge
at residues in equivalent positions near the Ca2+ coordination
site; Glu-454 in CBD1 is replaced by Lys-585 in CBD2. The unstructuring of
CBD1 upon Ca2+ removal was alleviated by reversing the charge of
the acidic residue (E454K) involved in Ca2+ coordination
(6). Previously, we solved the
structures of the Ca2+-bound and -free conformations of CBD2 and
revealed a charge compensation mechanism involving Lys-585
(4). The positively charged
lysine residue assumes the position of one of the Ca2+ ions upon
Ca2+ depletion, permitting CBD2 to retain its overall fold
(4). A similar phenomenon is
predicted to take place in E454K-CBD1 mutant. In addition, Hilge et
al. (6) showed that the
E454K mutation of CBD1 decreases Ca2+ affinity to a level similar
to that of CBD2 and suggested that the E454K mutation would cause the loss of
primary regulation of NCX1.1 by CBD1.The significance of some of these observations is unclear as a recent NMR
study (7) of CBD1 under more
physiologically relevant conditions revealed no significant alteration in
tertiary structure in the absence of Ca2+. It was hypothesized that
Ca2+ binding induces localized conformational and dynamic changes
involving several of the binding site residues. To clarify this issue, we
solved the crystal structure of the E454K-CBD1 mutant and examined the
functional effects of different CBD1 mutations in the full-length NCX1.1. The
results indicate that charge compensation is indeed provided by the residue
Lys-454 to replace one Ca2+, whereas the overall E454K-CBD1
structure is only slightly perturbed. The charge compensation, however, has no
impact on Ca2+ regulation of NCX1.1. 相似文献
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Nitric oxide synthase (NOS) plays a major role in a number of key physiological and pathological processes. Knowledge of how this is regulated is important. The small acidic calcium binding protein, calmodulin (CaM), is required to fully activate the enzyme. The exact mechanism of how CaM activates NOS is not fully understood. Studies have shown CaM to act like a switch that causes a conformational change in NOS to allow for the transfer of an electron between the reductase and oxygenase domains through a process that is thought to be highly dynamic. To investigate the dynamic properties of CaM-NOS interactions, we determined the solution structure of CaM bound to the inducible NOS (iNOS) and endothelial NOS (eNOS) CaM binding region peptides. In addition, we investigated the effect of CaM phosphorylation. Tyrosine 99 (Y99) of CaM is reported to be phosphorylated in vivo. We have produced a phosphomimetic Y99E CaM to investigate the structural and functional effects that the phosphorylation of this residue may have on nitric oxide production. All three mammalian NOS isoforms were included in the investigation. Our results show that a phosphomimetic Y99E CaM significantly reduces the maximal synthase activity of eNOS by 40% while having little effect on nNOS or iNOS activity. A comparative nuclear magnetic resonance study between phosphomimetic Y99E CaM and wild-type CaM bound to the eNOS CaM binding region peptide was performed. This investigation provides important insights into how the increased electronegativity of a phosphorylated CaM protein affects the binding, dynamics, and activation of the NOS enzymes. 相似文献