Role Of Methionine Research Articles

A role of methionines in the functioning of oxidatively modified fibrinogen

Posttranslational modifications in fibrinogen resulting from induced oxidation or oxidative stress in the organism can have deleterious influence on optimal functioning of fibrinogen, causing a disturbance in assembly and properties of fibrin. The protective mechanism supporting the ability of fibrinogen to function in ROS-generating environment remains completely unexplored. The effects of very low and moderately low HOCl/−OCl concentrations on fibrinogen oxidative modifications, the fibrin network structure as well as the kinetics of both fibrinogen-to-fibrin conversion and fibrin hydrolysis have been explored in the current study. As opposed to 25 Μm, HOCl/−OCl, 10 μM HOCl/−OCl did not affect the functional activity of fibrinogen. It is shown for the first time that a number of Met residues, AαMet476, AαMet517, AαMet584, BβMet367, γMet264, and γMet94, identified in 10 μM HOCl/–OCl fibrinogen by the HPLC-MS/MS method, operate as ROS scavengers, performing an important antioxidant function. In turn, this indicates that the fibrinogen structure is adapted to the detrimental action of ROS. The results obtained in our study provide evidence for a protective mechanism responsible for maintaining the structure and functioning of fibrinogen molecules in the bloodstream under conditions of mild and moderate oxidative stress.

Neurocritical Care Nutrition: Unique Considerations and Strategies for Optimizing Energy Supply and Metabolic Support in Critically Ill Patients

Neuro-critically ill patients face unique challenges in nutritional support due to varying metabolic and energy demands arising from clinical situations such as elevated intracranial pressure, trauma, stroke, epilepsy, targeted temperature management (TTM), and pharmacological interventions. Despite these challenges, there is a lack of systematic reviews addressing their specific needs. This study conducts a systematic review and narrative synthesis of the existing literature on nutritional support for neuro-critical patients, focusing on amino acids that act as neurotransmitters or their precursors. We examined nutritional requirements and timing in patients with stroke and traumatic brain injury, as well as energy and metabolic demands during TTM, a common therapeutic intervention in neuro-critical care. Our review aims to clarify uncertainties surrounding amino acid provision in neuro-critical patients and provide up-to-date recommendations on nutritional support for those with elevated intracranial pressure and undergoing TTM, serving as a foundation for future research and evidence-based guideline development. Key findings include the potential benefits of branched-chain amino acids (BCAAs) in neuro-critical care, the roles of methionine and serine, the significance of arginine in vascular constriction, the controversial role of glutamate in nutrition, and the potential benefits of selenium supplementation. We also discussed special considerations in nutritional support for neuro-critical patients, such as changes in energy demand, the influence of sedation on energy demand, and metabolism changes during TTM. Our review highlights the need for a better understanding of the unique metabolic requirements of neuro-critical patients and the development of evidence-based guidelines for optimal nutritional support in this population.

Integrated analysis of the association between methionine cycle and risk of moyamoya disease.

The role of methionine (Met) cycle in the pathogenesis and progression of cardiovascular and cerebrovascular diseases has been established, but its association with moyamoya disease (MMD) has rarely been studied. This study aimed to analyze the levels of Met cycle-related metabolites and constructed a risk model to explore its association with the risk of MMD. In this prospective study, a total of 302 adult MMD patients and 88 age-matched healthy individuals were consecutively recruited. The serum levels of Met cycle-related metabolites were quantified by liquid chromatography-mass spectrometry (LC-MS). Participants were randomly divided into training set and testing set at a ratio of 1:1. The training set was used to construct the risk score model by LASSO regression. The association between Met cycle-related risk score and the risk of MMD was analyzed using logistic regression and assessed by ROC curves. The testing set was used for validation. The levels of methionine sulfoxide and homocysteine were significantly increased, while the levels of betaine and choline were significantly decreased in MMD and its subtypes compared to healthy controls (p < 0.05 for all). The training set was used to construct the risk model and the risk score of each participant has been calculated. After adjusting for potential confounders, the risk score was independently associated with the risk of MMD and its subtypes (p < 0.05 for all). We then divided the participants into low-risk and high-risk groups, the high-risk score was significantly associated with the risk of MMD and its subtypes (p < 0.05 for all). The risk scores were further assessed as tertiles, the highest tertile was significantly associated with a higher risk of MMD and its subtypes compared to the lowest (p < 0.05 for all). The results were validated in the testing set. This study has constructed and validated a risk model based on Met cycle-related metabolites, which was independently associated with the risk of MMD and its subtypes. The findings provided a new perspective on the risk evaluation and prevention of MMD.

Roles of methionine and cysteine residues of the Escherichia coli sensor kinase HprS in reactive chlorine species sensing.

Sensor histidine kinase HprS, an oxidative stress sensor of Escherichia coli, senses reactive oxygen species (ROS) and reactive chlorine species (RCS), and is involved in the induction of oxidatively damaged protein repair periplasmic enzymes. We reinvestigated the roles of six methionine and four cysteine residues of HprS in the response to HClO, an RCS. The results of site-directed mutagenesis revealed that methionine residues in periplasmic and cytoplasmic regions (Met225) are involved in HprS activation. Interestingly, the Cys165Ser substitution reduced HprS activity, which was recovered by an additional Glu22Cys substitution. Our results demonstrate that the position of the inner membrane cysteine residues influences the extent of HprS activation in HClO sensing.

Abstract SY41-03: Cancer specific amino acid metabolism

Abstract The growth &amp; proliferation of cancer cells is fundamentally dependent on metabolic processes, and tumours frequently display altered metabolism compared with healthy tissues. To sustain enhanced growth, and to resist stresses associated with cancer treatments, cancer cells become highly dependent on the uptake of exogenous nutrients, particularly amino acids. Amino acid metabolism supports tumour growth and survival in numerous ways, particularly in the provision of anabolic precursors for macromolecule (e.g. protein and DNA) synthesis, as well as stress responsive antioxidant pathways (e.g. glutathione/GSH synthesis). We have shown that uptake of exogenous serine and glycine is key nutrient dependency for many cancer cells, simultaneously supporting THF-dependent one carbon metabolism and GSH synthesis. More recently we have defined a role for immune-regulated IDO1 dependent tryptophan metabolism as an alternative one-carbon source for nucleotide synthesis, underlining the potential flexibility of tumour metabolism. The sensitivity of certain cancer cells to cysteine starvation is well established, and multiple mechanisms contribute to cysteine withdrawal sensitivity, including transsulfuration activity and methylation reactions. We have recently defined a role for methionine driven polyamine metabolism in sensitising cells to cysteine starvation, particularly in the context of MTAP gene deletion. Improved understanding of cancer specific amino acid metabolism is defining genetically driven metabolic vulnerabilities that can be therapeutically exploited by a combination of diet and small molecules. Citation Format: Oliver Maddocks. Cancer specific amino acid metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr SY41-03.

Rice protein suppresses 4-hydroxy-2-nonenal-induced inflammation owing to methionine availability.

4-Hydroxy-2-nonenal (HNE) is one of the most important products of lipid peroxidation, which induces inflammation. To investigate the effects of rice protein (RP) on suppressing HNE-induced inflammation and the role of methionine in regulating the anti-inflammatory function of RP, Wistar rats (male, weighing 180-200 g) were either ad libitum fed a pellet diet with oral administration of methionine or ad libitum fed RP for 2 weeks. RP and methionine significantly reduced HNE levels and effectively suppressed the expressions of cyclooxygenase-2, tumor necrosis factor alpha, interleukin-1β (IL-1β) and IL-6, and inducible nitric oxide synthase. The anti-inflammatory action of RP was evident from the upregulation of IL-10 and glutathione S-transferase (GST), which played a role in the detoxification of HNE. The results show that the molecular mechanism responsible for the anti-inflammatory function of RP is the inhibition of nuclear factor kappa B (NF-κB) activation by the downregulation of protein kinase B/phosphoinositide 3 kinase. Further, this study demonstrates that Met availability contributes to the suppression of HNE-induced inflammation through upregulating IL-10 and GST in rats fed RP. Novelty: RP suppresses HNE-induced inflammation. Methionine plays a role in upregulating IL-10 and GST. Methionine availability regulates the inhibition of NF-κB by RP.

On the roles of methionine and the importance of its microenvironments in redox metalloproteins.

The amino acid residue methionine (Met) is commonly thought of as a ligand in redox metalloproteins, for example in cytochromes c and in blue copper proteins. However, the roles of Met can go beyond a simple ligand. The thioether functional group of Met allows it to be considered as a hydrophobic residue as well as one that is capable of weak dipolar interactions. In addition, the lone pairs on sulphur allow Met to interact with other groups, inluding the aforementioned metal ions. Because of its properties, Met can play diverse roles in metal coordination, fine tuning of redox reactions, or supporting protein structures. These roles are strongly influenced by the nature of the surrounding medium. Herein, we describe several common interactions between Met and surrounding aromatic amino acids and how they affect the physical properties of both copper and iron metalloproteins. While the importance of interactions between Met and other groups is established in biological systems, less is known about their roles in redox metalloproteins and our view is that this is an area that is ready for greater attention.

Methionine represses the autophagy of gastric cancer stem cells via promoting the methylation and phosphorylation of RAB37

ABSTRACT This study focused on the role of methionine (MET) in the autophagy of gastric cancer stem cells (GCSCs) and aims to elaborate its regulatory mechanism. In the present study, the GCSCs were isolated from human gastric cancer cell lines using an anti-CD44 antibody, and then cultured in MET+ homocysteine (HCY)− or MET−HCY+ medium. In MET+HCY–treated GCSCs, autophagy was suppressed, the methylation and phosphorylation of RAB37 were elevated, and miR-200b expression was down-regulated. Lentiviral vector (LV-) carrying methionine-γ lyase (an enzyme that could specifically lyse MET; Metase) promoted autophagy, reduced the methylation and phosphorylation of RAB37, and up-regulated miR-200b expression in MET+HCY–-treated GCSCs. Then, we found that miR-200b suppressed the expression of protein kinase C α (PKCα), a protein that could inactivate RAB37 through promoting its phosphorylation. LV-Metase down-regulated RAB37 phosphorylation via miR-200b/PKCα, thus promoting the RAB37-mediated autophagy and suppressing cell viability in MET+HCY–treated GCSCs. Finally, the in vivo study proved that LV-Metase treatment inhibited tumor growth through up-regulating RAB37 expression. In conclusion, MET suppressed RAB37 expression via enhancing its methylation and suppressed RAB37 activity via miR-200b/PKCα axis, thus repressing RAB37-mediated autophagy in GCSCs. The supplementation of Metase lysed MET, thus inducing the autophagy of GCSCs and inhibiting tumor growth.

Role of methionine in the correction of oxidative stress with increased physical exertion

Methionine is an indispensable sulfur-containing amino acid that is involved in processes unique to the body: initiation of protein synthesis; methylation process, an adenosine residue is attached to methionine and S-adenosylmethionine is formed; as a result of metabolic reactions, this methyl group is transferred to a number of substrates. In this case, adrenaline, melatonin, creatine, and other amino acids are formed. This article contains a literature review of articles that prove the antioxidant properties of methionine on the cell and the body as a whole. A large amount of literature and databases, such as Pubmed, Google.Scholar, Embase, have been worked out to search for information.

Methionine in proteins: The Cinderella of the proteinogenic amino acids.

Methionine in proteins, apart from its role in the initiation of translation, is assumed to play a simple structural role in the hydrophobic core, in a similar way to other hydrophobic amino acids such as leucine, isoleucine, and valine. However, research from a number of laboratories supports the concept that methionine serves as an important cellular antioxidant, stabilizes the structure of proteins, participates in the sequence-independent recognition of protein surfaces, and can act as a regulatory switch through reversible oxidation and reduction. Despite all these evidences, the role of methionine in protein structure and function is largely overlooked by most biochemists. Thus, the main aim of the current article is not so much to carry out an exhaustive review of the many and diverse processes in which methionine residues are involved, but to review some illustrative examples that may help the nonspecialized reader to form a richer and more precise insight regarding the role-played by methionine residues in such processes.

Methionine supplementation stimulates mitochondrial respiration

Mitochondria play essential metabolic functions in eukaryotes. Although their major role is the generation of energy in the form of ATP, they are also involved in maintenance of cellular redox state, conversion and biosynthesis of metabolites and signal transduction. Most mitochondrial functions are conserved in eukaryotic systems and mitochondrial dysfunctions trigger several human diseases.By using multi-omics approach, we investigate the effect of methionine supplementation on yeast cellular metabolism, considering its role in the regulation of key cellular processes. Methionine supplementation induces an up-regulation of proteins related to mitochondrial functions such as TCA cycle, electron transport chain and respiration, combined with an enhancement of mitochondrial pyruvate uptake and TCA cycle activity. This metabolic signature is more noticeable in cells lacking Snf1/AMPK, the conserved signalling regulator of energy homeostasis. Remarkably, snf1Δ cells strongly depend on mitochondrial respiration and suppression of pyruvate transport is detrimental for this mutant in methionine condition, indicating that respiration mostly relies on pyruvate flux into mitochondrial pathways.These data provide new insights into the regulation of mitochondrial metabolism and extends our understanding on the role of methionine in regulating energy signalling pathways.

Upstream regulators of apoptosis mediates methionine-induced changes of lipid metabolism

Although the role of methionine (Met), as precursor for l-carnitine synthesis, in the regulation of lipid metabolism has been explored. Met seems to have tissue- and species-specific regulatory effect on lipid metabolism, implying that the mechanisms in Met regulation of lipid metabolism is complex and may involve the upstream regulatory pathway of lipid metabolism. The present study was performed to determine the mechanism of apoptosis signaling pathways mediating Met-induced changes of hepatic lipid deposition and metabolism in fish, and compare the differences of the mechanisms between the fish and mammals. By iTRAQ-based quantitative proteome analyses, we found that both dietary Met deficiency and excess evoked apoptosis signaling pathways, increased hepatic lipid deposition and caused aberrant hepatic lipid metabolism of yellow catfish Pelteobagrus fulvidraco. Using primary hepatocytes from P. fulvidraco, inhibition of caspase by Z-VAD-FMK blocked the apoptotic signaling pathways with a concomitant reversal of Met deficiency- and excess-induced increase of lipid deposition, indicating that apoptosis involved the Met-mediated changes of hepatic lipid metabolism. Moreover, we explored the roles of three upstream apoptotic signaling pathways (PI3K/AKT-TOR pathway, cAMP/PKA/CREB pathway and LKB1/AMPK-FOXO pathway) influencing hepatic lipid metabolism of P. fulvidraco. The three upstream pathways participated in apoptosis mediating Met-induced changes of lipid metabolism in P. fulvidraco. At last, HepG2 cell line was used to compare the similarities of mechanisms in apoptosis mediating Met-induced changes of lipid metabolism between fish and mammals. Although several slight differences existed, apoptosis mediated the Met-induced changes of lipid metabolism between fish and mammals. The present study reveals novel apoptosis-relevant signal transduction axis which mediates the Met-induced changes of lipid metabolism, which will help understand the mechanistic link between apoptosis and lipid metabolism, and highlight the importance of the evolutionary conservative apoptosis signaling axis in regulating Met–induced changes of hepatic lipid metabolism.

The Role of Calmodulin Methionine Oxidation in Regulating Conformational Change

We have used molecular dynamics simulations to understand the role of calmodulin (CaM) methionine (Met) oxidation in disrupting calcium-induced conformational changes associated with CaM's regulation of its target proteins. Previous work suggests that C-terminal CaM methionines, particularly M109 and M124, are critical for functional interaction with the major calcium release channel in muscle, the ryanodine receptor (RyR). Methionine to glutamine (Gln) substitutions at M109 or M124 designed to mimic oxidation of Met to Met sulfoxide block CaM activation of RyR at low calcium levels and inhibition of RyR at high calcium levels (Journal of Biological Chemistry 278(18) 2003). Spectroscopic distance measurements across CaM's lobes suggest that these same Met to Gln substitutions altered the equilibrium between key CaM conformations (Biochemical and biophysical research communications 456(2) 2015). We carried out microsecond molecular dynamics simulations of the open and closed CaM structural states with Met to Gln substitutions at M109 or M124 to gain an atomic-level understanding of (1) the role of methionines in stabilizing CaM structure, and (2) how Met oxidation might influence conformational equilibria and regulatory interactions. We found that every CaM C-terminal Met participates in a Met-aromatic interaction with nearby Phe residues with interaction energies of ∼4-10 kcal/mol. M109 stably interacts with F92, but only in the open structure, and M124 stably interacts with F141, but only in the closed structure. Met to Gln substitution at M109 or M124 alters interaction energies with these nearby Phe and alters the stability of the open and closed structures. We expect that these results will be applicable to understanding the general mechanism through which Met oxidation orchestrates redox cell signaling.

The role of methionine on metabolism, oxidative stress, and diseases.

Methionine is an aliphatic, sulfur-containing, essential amino acid, and a precursor of succinyl-CoA, homocysteine, cysteine, creatine, and carnitine. Recent research has demonstrated that methionine can regulate metabolic processes, the innate immune system, and digestive functioning in mammals. It also intervenes in lipid metabolism, activation of endogenous antioxidant enzymes such as methionine sulfoxide reductase A, and the biosynthesis of glutathione to counteract oxidative stress. In addition, methionine restriction prevents altered methionine/transmethylation metabolism, thereby decreasing DNA damage and carcinogenic processes and possibly preventing arterial, neuropsychiatric, and neurodegenerative diseases. This review focuses on the role of methionine in metabolism, oxidative stress, and related diseases.

Role of methionine on epigenetic modification of DNA methylation and gene expression in animals

DNA methylation is one of the main epigenetic phenomena affecting gene expression. It is an important mechanism for the development of embryo, growth and health of animals. As a key nutritional factor limiting the synthesis of protein, methionine serves as the precursor of S-adenosylmethionine (SAM) in the hepatic one-carbon metabolism. The dietary fluctuation of methionine content can alter the levels of metabolic substrates in one-carbon metabolism, e.g., the SAM, S-adenosylhomocysteine (SAH), and change the expression of genes related to the growth and health of animals by DNA methylation reactions. The ratio of SAM to SAH is called ‘methylation index’ but it should be carefully explained because the complexity of methylation reaction. Alterations of methylation in a specific cytosine-guanine (CpG) site, rather than the whole promoter region, might be enough to change gene expression. Aberrant methionine cycle may provoke molecular changes of one-carbon metabolism that results in deregulation of cellular hemostasis and health problems. The importance of DNA methylation has been underscored but the mechanisms of methionine affecting DNA methylation are poorly understood. Nutritional epigenomics provides a promising insight into the targeting epigenetic changes in animals from a nutritional standpoint, which will deepen and expand our understanding of genes, molecules, tissues, and animals in which methionine alteration influences DNA methylation and gene expression.

From yeast to human: exploring the comparative biology of methionine restriction in extending eukaryotic life span.

Methionine restriction is a widely reported intervention for increasing life span in several model organisms. Low circulating levels of methionine are evident in the long-lived naked mole-rat, suggesting that it naturally presents with a life-extending phenotype akin to that observed in methionine-restricted animals. Similarly, long-lived dwarf mice also appear to have altered methionine metabolism. The mechanisms underlying methionine-restriction effects on life-span extension, however, remain unknown, as do their potential connections with caloric restriction, another well-established intervention for prolonging life span. Paradoxically, methionine is enriched in proteins expressed in mitochondria and may itself serve an important role in the detoxification of reactive oxygen species and may thereby contribute to delayed aging. Collectively, we highlight the evidence that modulation of the methionine metabolic network can extend life span-from yeast to humans-and explore the evidence that sulfur amino acids and the concomitant transsulfuration pathway play a privileged role in this regard. However, systematic studies in single organisms (particularly those that exhibit extreme longevity) are still required to distinguish the fundamental principles concerning the role of methionine and other amino acids in regulating life span.

Role of methionine containing antioxidant combination in the management of pain in chronic pancreatitis: A systematic review and meta-analysis

BackgroundPain in CP results from inflammation and neuroimmune alterations that are associated with oxidative stress, among other mechanisms. This is marked by depletion of antioxidant defenses including methionine, which is a donor of methyl moieties that maintains the acinar transsulfuration pathway. We performed a systematic review and meta-analysis of trials evaluating methionine-containing antioxidants in CP. Patient and methodsLiterature search was conducted in Medline/Pubmed, EMBASE, and Cochrane databases. Systematic review and meta-analysis was performed per PRISMA guidelines. Main study outcome was pain relief. GRADE system was used for quality assessment. Heterogeneity was assessed by the Q and I2 measures; publication bias by Egger's test. Random-effect model (DerSimonian and Laird) was used if there was heterogeneity. ResultsEight studies (n = 411) were identified that used methionine-containing antioxidants. The study duration ranged from 10 wks to 12 months. All studies used methionine, organic selenium, ascorbate, beta-carotene and alpha-tocoferol. Four studies (including two RCTs) that reported change in pain scores were metaanalyzed. Though overall effect [standardized difference in means (95% CI)] on pain score reduction was −0.95 (−1.738 to −0.160) (z = −2.36; p = 0.018), the significance was lost when the two RCTs were meta-analyzed. RCTs that reported the number of pain free patients had a statistically significant overall effect of −3.204 (p = 0.001). Though more patients on methionine containing antioxidants had adverse events, majority of them were mild. ConclusionMethionine containing antioxidants appear to result in pain reduction in a significant proportion of CP patients. Further randomized controlled trials with homogeneous outcome measures are needed.

Abstract TP79: Imbalance Between Activation Of Matrix Metalloprotease And Its Inhibition May Facilitate The Growth Of Cerebral Aneurysm In Female Rats

Introduction: Cerebral aneurysms are a major cause of subarachnoid hemorrhage . Despite the catastrophic consequences of their rupture, not all mechanisms underlying the formation, progression, and rupture of cerebral aneurysms are fully understood. Using our model of reproducible cerebral aneurysms in oophorectomized rats we have documented the pathogenesis of cerebral aneurysms. Methionine-induced hyperhomocysteinemia which participates in coronary disease or stroke, is also associated with cerebral aneurysm formation. Whether it facilitates the growth and rupture of cerebral aneurysms remains obscure. We explored the role of methionine in the growth and rupture of cerebral aneurysms. Methods: We subject 13 week-old female Sprague-Dawley rats fed an 8% high-salt diet to bilateral oophorectomy and ligation of the right common carotid artery. Four weeks later they underwent ligation of the bilateral posterior renal artery divided into 2 groups; one group did and the other did not receive 1g/kg/day methionine in the drinking water. The aneurysmal rupture rate and morphologic- and genomic changes in the 2 groups were compared at 12 weeks. Results: The incidence of cerebral aneurysm formation was the same in both groups. While the rate of rupture was not higher in the methionine group, their aneurysms were larger and oxidative stress-related NOX4, Rac1, and apoptosis-related Rac1 were up-regulated. Interestingly, the expression of MMP-9 was increased as was the ratio of MMP-9 to TIMP2 but not of MMP-9 to TIMP1, suggesting vascular degradation due to an imbalance between the activation and inhibition of extracellular matrix metalloprotease. Conclusion: Our findings suggest that excessive vascular degradation due to oxidative stress and apoptosis induced by methionine contribute to the progression of cerebral aneurysms in oophorectomized rats.

Thermodynamic and spectroscopic investigation on the role of Met residues in CuII binding to the non-octarepeat site of the human prion protein

Among the common features shared by neurodegenerative diseases there is the central role played by specific proteins or peptides which accumulate in neurons as insoluble plaques or tangles, containing abnormal amounts of redox-active metal ions, like copper and iron. In the case of transmissible spongiform encephalopathies (TSE), the involved protein is known as "prion protein" (PrP(C)) since "prions" (proteinaceous and infectious) are the agents which make TSE transmissible. It is widely accepted that PrP(C), in its wild-type form, can bind up to six Cu(II) ions, four of them in the so-called "octarepeat domain" and the others in the "fifth (non-octarepeat) binding-site". The latter domain contains two His residues, acting as anchoring sites for Cu(II) ions, and other potential binding residues, such as Lys and Met. While it is widely accepted that Lys residues do not take part in complex-formation, the role of methionines is still debated. In order to shed light on this issue, some peptides have been synthesized, either directly mimicking the sequence of the second half of the fifth binding site of human-PrP(C) (apo-form) or analogues where Met residues have been substituted by n-leucine. In addition, a series of short peptides, containing both His and Met residues in different relative positions, have been investigated, for the sake of comparison. Spectroscopic results, including NMR spectra of systems containing Ni(II) as a probe for the paramagnetic Cu(II) ion, agree on the exclusion of any direct interaction between the sulphur atom of Met residues and the Cu(II) ion already bound to His-imidazole side-chains. However, thermodynamic data show that Met-109 somewhat contributes to stability of complex species and this can be attributed to different electronic and steric effects.

Fast methionine-based solution structure determination of calcium-calmodulin complexes

Here we present a novel NMR method for the structure determination of calcium-calmodulin (Ca(2+)-CaM)-peptide complexes from a limited set of experimental restraints. A comparison of solved CaM-peptide structures reveals invariability in CaM's backbone conformation and a structural plasticity in CaM's domain orientation enabled by a flexible linker. Knowing this, the collection and analysis of an extensive set of NOESY spectra is redundant. Although RDCs can define CaM domain orientation in the complex, they lack the translational information required to position the domains on the bound peptide and highlight the necessity of intermolecular NOEs. Here we employ a specific isotope labeling strategy in which the role of methionine in CaM-peptide interactions is exploited to collect these critical NOEs. By (1)H, (13)C-labeling the methyl groups of deuterated methionine against a (2)H, (12)C background, we can acquire a (13)C-edited NOESY characterized by simplified, easily analyzable spectra. Together with measured CaM backbone H(N)-N RDCs and intrapeptide NOE-based distances, these intermolecular NOEs provide restraints for a low temperature torsion-angle dynamics and simulated annealing protocol used to calculate the complex structure. We have applied our method to a CaM complex previously solved through X-ray crystallography: Ca(2+)-CaM bound to the CaM kinase I peptide (PDB code: 1MXE). The resulting structure has a backbone RMSD of 1.6 Å to that previously published. We have also used this test complex to investigate the importance of homologous model selection on the calculated outcome. In addition to having application for fast complex structure determination, this method can be used to determine the structures of difficult complexes characterized by chemical shift overlap and broad signals for which the traditional method based on the use of fully (13)C, (15)N-labeled CaM fails.