Psychological distress and need for psycho-oncological support in spouses of total laryngectomised cancer patients-results for the first 3 years after surgery.

PURPOSE: A total laryngectomy (TLE) leads to a variety of functional restrictions, which reduce the quality of life of cancer patients as well as their spouses. However, to date, there is little research focusing on the psychological distress of spouses of total laryngectomised cancer patients. The current study assesses psychological distress, need for psycho-oncological treatment and use of professional psychological care among spouses of total laryngectomised cancer patients. METHODS: A prospective multi-centre cohort study was conducted. Participants were interviewed in person 1, 2 and 3 years subsequent to their spouses' TLE with standardised questionnaires (HADS, Hornheide Screening) and self-designed items. RESULTS: One year after their partners' TLE, 154 spouses were interviewed. Over half of spouses (57 %) reported a high level of psychological distress and 33 % reported restlessness. Majority of spouses (21 %) reported wanting to learn relaxation methods and eight (5 %) had received psychological treatment in the past. Sixty-two spouses took part in the complete study. Over all three time points, psychological distress, the need for psycho-oncological support and the use of professional support among spouses remained stable. The need for additional professional counselling was low. CONCLUSIONS: In view of the stability of psychological distress among half of the spouses within 3 years after TLE and their refusal of professional support, there is a need for the development and evaluation of new treatment strategies to help spouses cope with psychological distress. Our results indicated the most common additional professional need was learning relaxation methods, which may be used as a starting point for the investigation of new coping strategies in future studies.

Molecular dynamics simulations reveal that apo-HisJ can sample a closed conformation.

The Escherichia coli histidine binding protein HisJ is a type II periplasmic binding protein (PBP) that preferentially binds histidine and interacts with its cytoplasmic membrane ABC transporter, HisQMP2 , to initiate histidine transport. HisJ is a bilobal protein where the larger Domain 1 is connected to the smaller Domain 2 via two linking strands. Type II PBPs are thought to undergo "Venus flytrap" movements where the protein is able to reversibly transition from an open to a closed conformation. To explore the accessibility of the closed conformation to the apo state of the protein, we performed a set of all-atom molecular dynamics simulations of HisJ starting from four different conformations: apo-open, apo-closed, apo-semiopen, and holo-closed. The simulations reveal that the closed conformation is less dynamic than the open one. HisJ experienced closing motions and explored semiopen conformations that reverted to closed forms resembling those found in the holo-closed state. Essential dynamics analysis of the simulations identified domain closing/opening and twisting as main motions. The formation of specific inter-hinge strand and interdomain polar interactions contributed to the adoption of the closed apo-conformations although they are up to 2.5-fold less prevalent compared with the holo-closed simulations. The overall sampling of the closed form by apo-HisJ provides a rationale for the binding of unliganded PBPs with their cytoplasmic membrane ABC transporters.

[Social integration and its relevance for quality of life after laryngectomy]. / Soziale Integration und deren Bedeutung für die Lebensqualität nach Laryngektomie.

BACKGROUND: Social networks and social participation generally have positive effects on health. Yet, little is known about how patients after total laryngectomy (TLE) are integrated into the society. Aim of this study was to investigate how patients are socially integrated after a TLE and if social integration is associated with certain areas of quality of life. PATIENTS AND METHODS: In a longitudinal multi-centred study 161 laryngectomees were interviewed 1 year after the total laryngectomy. Social integration was measured on the basis of an index formed by the questionnaire "Psychosocial Adjustment after Laryngectomy" and questions about social support. To assess quality of life, we used the questionnaire from the European Organisation for Research and Treatment of Cancer EORTC QLQ-C30. RESULTS: 58% of all patients are well integrated 1 year after surgery. Well integrated persons have less problems in different components of quality of life. They report higher levels of social (OR 4.07; CI: 1.96-8.47) and role functioning (OR 3.59; CI: 1.61-8.02). Successful social integration is also associated with higher emotional well-being (OR 8.57; CI: 3.59-20.46). CONCLUSIONS: There is evidence that 1 year after TLE only about half of the patients feel socially integrated. Because of the negative association of poor social integration with social, emotional and role functioning, patients should be supported in their attempts to take actively part in social life.

Mental disorders and psychosocial support during the first year after total laryngectomy: a prospective cohort study.

OBJECTIVES: To assess the frequency of mental disorders and the use of psychosocial services in laryngectomised patients during the first year after surgery. DESIGN: Multicentre prospective study including six interviews. Data regarding psychiatric comorbidity 3 months (3 m) and 1 year (12 m) after total laryngectomy (TLE) are reported in this study. SETTING: Structured interviews were conducted at nine hospitals and three rehabilitation centres in Germany. PARTICIPANTS: One hundred and seventy-one patients were interviewed at both time-points. MAIN OUTCOME MEASURES: Structured clinical interview for DSM-IV (SCID). RESULTS: Mental disorders were diagnosed in 25% of the patients (3 m) and in 22% of the patients (12 m), respectively. Six per cent of the patients developed a mental disorder during the first year after total laryngectomy. In general, male and female patients suffered from mental disorders with equal frequency (3 m: 23% versus 37%; P = 0.26; 12 m: 22% versus 21%; P = 1.00). Women suffered more often than men from post-traumatic stress disorder (3 m) (P = 0.01) and generalised anxiety disorder (12 m) (P = 0.01).Of the patients who had acquired no voice, 20% suffered from alcohol dependence (P = 0.01) [corrected]. There were no differences between men and women in receiving any kind of counselling (P = 0.79) or psychotherapy/psychiatric treatment (P = 0.47). Of those patients diagnosed with any mental disorder 3 months after total laryngectomy, 7% had received psychotherapy 1 year after total laryngectomy. None of the patients diagnosed with alcohol dependence received psychotherapy or psychiatric treatment. CONCLUSIONS: Mental disorders occur in laryngectomees as frequently in men as they do in women. Total laryngectomised patients who were mentally ill did not receive enough psychotherapeutic or psychiatric support. As mental health seems to be related to successful voice restoration, future research should develop and evaluate special psychosocial supportive programmes for patients with laryngeal cancer, especially regarding alcohol dependence treatment.

[Use of cancer support groups by laryngectomees in central Germany]. / Inanspruchnahme von Selbsthilfegruppen für Laryngektomierte in Mitteldeutschland.

BACKGROUND: Cancer support groups provide information and coping resources as well as represent patients' interests. To date it is unknown how often cancer patients post-laryngectomy use support groups and in which parameters users of support groups differ from non-users. MATERIAL AND METHODS: In a multicentre study, 224 laryngectomees were asked about their support group membership. Further, possible predictors for membership one year post-surgery were assessed. Data were collected with a semi-structured interview and standardized instruments. RESULTS: Overall, 23% of the laryngectomized patients are actively involved in cancer support groups. The probability of a membership increases if patients are well-educated, are living in good economic conditions and in a partnership, if they perceive low family support and wish additional counselling with a physician. CONCLUSION: A cancer support group seems to "buffer" family support perceived to be insufficient. However, support group users are living more frequently in a partnership and in good economic conditions compared to non-users. Physicians and speech therapists are important mediators to cancer support groups. They particularly should inform laryngectomees who are living in bad economic conditions and who are not living in a partnership about the availability of cancer support groups.

[Vocational rehabilitation after total laryngectomy]. / Berufliche Rehabilitation nach Laryngektomie.

BACKGROUND: Aim of this study was to find out how many patients after a total laryngectomy (TLE) return to work successfully and what factors support vocational rehabilitation. PATIENTS AND METHODS: Laryngectomees (n=231) aged up to 60 years completed questionnaires and structured interviews before TLE (t1), before rehabilitation (t2), at the end of rehabilitation (t3), 1 year after TLE (t4), 2 years after TLE (t5), and 3 years after TLE (t6). RESULTS: Prior to TLE, 38% of all respondents were employed, 34% were unemployed, 23% received disability-related and 3% age-related pension retirement. One year after TLE, 13% were employed, 15% 2 years and 14% 3 years after TLE. Unemployed were 10% (t4), 5% (t5), and 7% (t6) of the patients. For 59% of all respondents it was very important to have a job. Predictors of successful vocational rehabilitation were employment prior to TLE, age <50 years, being self-employed or clerical employee, good physical functioning, good speech intelligibility, high motivation to go back to work, and support from colleagues. CONCLUSION: Only few laryngectomees return to work. However, even before TLE only a third of the patients was employed, another third was unemployed. Most of the patients receive pension retirement after TLE. As return to work is important for many patients, patient consultations should consider possibilities to support vocational rehabilitation before offering to apply for retirement.

Differentiating short- and long-term effects of diet in the obese mouse using (1) H-nuclear magnetic resonance metabolomics.

This study determined whether targeted metabolomic profiling of serum, using 1H nuclear magnetic resonance, could be employed to distinguish the effects of obesity from those of diet in mice. Following weaning, littermates were randomly divided into two diet groups: chow and high fat. After 12 weeks of dietary manipulation, fat-fed animals were obese and hyperglycaemic. Mice from each treatment either maintained their current diet or switched to the opposite diet for a final week. Differences in metabolite levels were determined using orthogonal projection to latent structures and cross-validated discriminant analysis. The short- and long-term effects of each diet could be clearly distinguished. Short-term diet effects are the major contributor to the metabolic profile, underscoring the need for controls beyond the standard fast before serum collection. This work shows the importance of dietary controls when attempting to isolate obesity-related changes and highlights the ability of metabolomics to identify subtle changes when experiments are properly structured.

Metabolomic profiling of dietary-induced insulin resistance in the high fat-fed C57BL/6J mouse.

The predictive ability of metabolic profiling to detect obesity-induced perturbations in metabolism has not been clearly established. Complex aetiologies interacting with environmental factors highlight the need to understand how specific manipulations alter metabolite profiles in this state. The aim of this study was to determine if targeted metabolomic profiling could be employed as a reliable tool to detect dietary-induced insulin resistance in a small subset of experimental animals (n = 10/treatment). Following weaning, male C57BL/6J littermates were randomly divided into two dietary groups: chow and high fat. Following 12 weeks of dietary manipulation, mice were fasted for 5 h prior to serum collection. The resultant high fat-fed animals were obese and insulin resistant as shown by a euglycaemic-hyperinsulinaemic clamp. Sera were analysed by proton nuclear magnetic resonance spectroscopy, and 46 known compounds were identified and quantified. Multivariate analysis by orthogonal partial least squares discriminant analysis, a projection method for class separation, was then used to establish models of each treatment. Models were able to predict class separation between diets with 90% accuracy. Variable importance plots revealed the most important metabolites in this discrimination to include lysine, glycine, citrate, leucine, suberate and acetate. These metabolites are involved in energy metabolism and may be representative of the perturbations taking place with insulin resistance. Results show metabolomics to reliably describe the metabolic effects of insulin resistance in a small subset of samples and are an initial step in establishing metabolomics as a tool to understand the biochemical signature of insulin resistance.

Lubricin/Proteoglycan 4 binds to and regulates the activity of Toll-Like Receptors In Vitro.

Proteoglycan 4 (PRG4/lubricin) is secreted by cells that reside in articular cartilage and line the synovial joint. Lubricin may play a role in modulating inflammatory responses through interaction with CD44. This led us to examine if lubricin could be playing a larger role in the modulation of inflammation/immunity through interaction with Toll-like receptors (TLRs). Human Embryonic Kidney (HEK) cells overexpressing TLRs 2, 4 or 5 and surface plasmon resonance were employed to determine if full length recombinant human lubricin was able to bind to and activate TLRs. Primary human synovial fibroblasts were also examined using flow cytometry and Luminex multiplex ELISA. A rat destabilization model of osteoarthritis (OA) was used to determine if lubricin injections were able to regulate pain and/or inflammation in vivo. Lubricin can bind to and regulate the activity of TLRs, leading to downstream changes in inflammatory signalling independent of HA. We confirmed these findings in vivo through intra-articular injections of lubricin in a rat OA model where the inhibition of systemic inflammatory signaling and reduction in pain were observed. Lubricin plays an important role in regulating the inflammatory environment under both homeostatic and tissue injury states.

Melatonin and serotonin interactions with calmodulin: NMR, spectroscopic and biochemical studies.

It has been reported that the hormone melatonin binds tightly to the ubiquitous calcium-regulatory protein, calmodulin (CaM) with a Kd value around 0.1 nM [Benítez-King et al., Biochim. Biophys. Acta, 1290 (1993) 191-196]. Normally CaM only binds to target proteins and various 20-residue synthetic peptides encompassing the CaM-binding domain of these target proteins with K(d) values ranging between 1.0 microM and 0.1 nM. Here we have studied the interaction of melatonin and several structurally related compounds--serotonin, 5-hydroxytryptophan, and tryptophan--to CaM through gel band shift assays, enzymatic competition assays with calcineurin, fluorescence spectroscopy, far and near UV circular dichroism spectropolarimetry and NMR spectroscopy. Fluorescence spectra show that the binding is calcium dependent. NMR studies with biosynthetically labelled methyl-13C-Met CaM show that melatonin and the other compounds interact with the hydrophobic cleft regions of the protein. Our NMR data show that melatonin binds to both domains of the dumbbell-shaped CaM, while serotonin appears to bind only to the C-terminal domain. This binding mode is further substantiated by fluorescence and gel band shift competition experiments with synthetic peptides from myosin light chain kinase and constitutive nitric oxide synthase. Circular dichroism spectra indicate that the secondary structure of CaM is not altered by addition of melatonin. Our data are internally consistent and reveal Kd values in the mM range for melatonin. Thus the binding of these compounds to CaM is substantially weaker than was previously reported and is unlikely to be of physiological significance.

Diversity of antimicrobial peptides and their mechanisms of action.

Antimicrobial peptides encompass a wide variety of structural motifs. Many peptides have alpha-helical structures. The majority of these peptides are cationic and amphipathic but there are also hydrophobic alpha-helical peptides which possess antimicrobial activity. In addition, some beta-sheet peptides have antimicrobial activity and even antimicrobial alpha-helical peptides which have been modified to possess a beta-structure retain part of their antimicrobial activity. There are also antimicrobial peptides which are rich in a certain specific amino acid such as Trp or His. In addition, antimicrobial peptides exist with thio-ether rings, which are lipopeptides or which have macrocyclic Cys knots. In spite of the structural diversity, a common feature of the cationic antimicrobial peptides is that they all have an amphipathic structure which allows them to bind to the membrane interface. Indeed, most antimicrobial peptides interact with membranes and may be cytotoxic as a result of disturbance of the bacterial inner or outer membranes. Alternatively, a necessary but not sufficient property of these peptides may be to be able to pass through the membrane to reach a target inside the cell. The interaction of these peptides with biological membranes is not just a function of the peptide but is also modulated by the lipid components of the membrane. It is not likely that this diverse group of peptides has a single mechanism of action, but interaction of the peptides with membranes is an important requirement for most, if not all, antimicrobial peptides.

NMR and stopped-flow studies of metal ion binding to alpha-lactalbumins.

1H-NMR spectroscopy and stopped-flow techniques have been used to investigate the binding of a host of metal ions to alpha-lactalbumins from bovine, goat, and human sources. We have identified two 1H-NMR markers diagnostic of metal ion binding to the high-affinity Ca2+-binding site of bovine alpha-lactalbumin, namely the signals corresponding to the delta-CH3 groups of Met-90, and a leucine, tentatively assigned to Leu-96. A number of metal ions other than Ca2+ bind to this site in either slow (La3+, Lu3+, Y3+, Sr2+, Sc3+) or fast (Cd2+, Ba2+, Pb2+) exchange. From competition experiments using this approach, we have determined an affinity series for metal ion binding at this site, in which lanthanides and Y3+ bind the strongest (Y3+>La3+, Lu3+>Ca2+>Sr2+>Cd2+, Pb2+, Ba2+>Sc3+). Several metal ions do not alter the 1H spectrum of bovine alpha-lactalbumin, retaining the protein in an 'apo-like' state. Evidence is given to support the notion that the paramagnetic divalent metal ions Co2+ and Cu2+ bind to a second distinct site, termed the 'zinc site', and that His-68 is involved in metal ion coordination. Finally, stopped-flow techniques using the indicator Xylenol orange were employed to obtain lanthanide off-rates for bovine, human, and goat alpha-lactalbumins (bovine and goat alpha-LA: k(off)(s-1) approximately 0.2 to 0.01 from La3+ to Lu3+; human alpha-LA: k(off)(s-1) approximately 0.02 to 0.001 from La3+ to Lu3+). In each case, we found that k(off) values decreased by an order of magnitude across the series, meaning that the dissociation constants for these metal ions are relatively constant. Data for the bovine and goat proteins are virtually identical, while the off-rates for human alpha-lactalbumin are appreciably slower. In addition, these rates are much slower than the Ca2+ off-rate for the bovine protein (k(off)(s-1) approximately 2 to 5), determined using the fluorescent indicator, BAPTA.

Two-dimensional NMR studies of selenomethionyl calmodulin.

Calmodulin (CaM) is a ubiquitous calcium regulatory protein that can interact with almost 30 different target proteins. The majority of the CaM-binding domains of the target proteins are believed to interact with two hydrophobic surfaces on Ca(2+)-CaM; these two regions are very rich in Met residues. To obtain more information about the role of these residues, we have biosynthetically incorporated selenomethionine (SeMet) in place of the nine Met residues of CaM. Amino acid analysis shows that the SeMet-CaM contains 15% Met and 85% SeMet. SeMet-CaM retains many of the properties of the wild-type protein; it activates the enzyme cyclic nucleotide phosphodiesterase, it binds to phenyl-Sepharose and myosin light chain kinase (MLCK) in a calcium-dependent manner, and it experiences a calcium-dependent band shift during SDS-gel electrophoresis. Moreover, by comparing the natural abundance (1H,13C)-heteronuclear multiple quantum coherence (HMQC) spectra of the calcium, apo and target peptide-bound forms of wild-type CaM and SeMet-CaM, we have found that the two proteins have very similar, if not identical, structures. Thus, the substitution of SeMet for Met does not cause a change in the conformation and function of CaM, in agreement with the results obtained for other proteins. The apo, calcium and target peptide-bound forms of SeMet-CaM were subsequently studied by natural abundance (1H,77Se)-heteronuclear multiple bond correlation (HMBC) and (1H,13C)-HMQC NMR. Nine well-resolved 77Se resonances could be observed. Substitution of SeMet for Met gave rise to the same 1H and 13C chemical shift changes for each individual Met residue, this facilitated making the assignments from known 1H,13C assignments of the Met residues. Some of these assignments were confirmed by studying Met-->Leu mutants of CaM. With the exception of Met76, which always remains solvent exposed, all resonances experienced large 77Se chemical shift changes upon the addition of Ca2+ and the MLCK peptide. The large shift changes indicate that the electron distribution in the SeMet side-chain can be adjusted for the different states of CaM, suggesting that the polarizability of sulfur or selenium may be important for the proper functioning of CaM. This study also shows that the natural abundance (1H,77Se)-HMBC experiment provides a sensitive approach for the study of SeMet proteins.

Peptide and metal ion-dependent association of isolated helix-loop-helix calcium binding domains: studies of thrombic fragments of calmodulin.

Calmodulin (CaM), the ubiquitous, eukaryotic, bilobal calcium-binding regulatory protein, has been cleaved by thrombin to create two fragments. TM1 (1-106) and TM2 (107-148). NMR and CD results indicate that TMI and TM2 can associate in the presence of Ca2+ to form a complex similar to native CaM, even though the cleavage site is not in the linker region between two helix-loop-helix domains, but rather within an alpha-helix. Cadmium-113 NMR results show that this complex has enhanced metal-ion binding properties when compared to either TM1 or TM2 alone. This complex can bind several CaM-binding target peptides, as shown by gel bandshift assays, circular dichroism spectra, and 13C NMR spectra of biosynthetically methyl-13C-Met-labeled TM1 and TM2; moreover, gel bandshift assays show that the addition of a target peptide strengthens the interactions between TM1 and TM2 and increases the stability of the complex. Cadmium-113 NMR spectra indicate that the TM1:TM2 complex can also bind the antipsychotic drug trifluoperazine. However, in contrast to CaM:peptide complexes, the TM1:TM2:peptide complexes are disrupted by 4 M urea; moreover, TM1 and TM2 in combination are unable to activate CaM-dependent enzymes. This suggests that TM1:TM2 mixtures cannot bind target molecules as tightly as intact CaM, or perhaps that binding occurs but additional interactions with the target enzymes that are necessary for proper activation are perturbed by the proteolytic cleavage. The results presented here reflect the importance of the existence of helix-loop-helix Ca2+-binding domains in pairs in proteins such as CaM, and extend the understanding of the association of such domains in this class of proteins in general.

Substitution of the methionine residues of calmodulin with the unnatural amino acid analogs ethionine and norleucine: biochemical and spectroscopic studies.

Calmodulin (CaM) is a 148-residue regulatory calcium-binding protein that activates a wide range of target proteins and enzymes. Calcium-saturated CaM has a bilobal structure, and each domain has an exposed hydrophobic surface region where target proteins are bound. These two "active sites" of calmodulin are remarkably rich in Met residues. Here we have biosynthetically substituted (up to 90% incorporation) the unnatural amino acids ethionine (Eth) and norleucine (Nle) for the nine Met residues of CaM. The substituted proteins bind in a calcium-dependent manner to hydrophobic matrices and a synthetic peptide, encompassing the CaM-binding domain of myosin light-chain kinase (MLCK). Infrared and circular dichroism spectroscopy show that there are essentially no changes in the secondary structure of these proteins compared to wild-type CaM (WT-CaM). One- and two-dimensional NMR studies of the Eth-CaM and Nle-CaM proteins reveal that, while the core of the proteins is relatively unaffected by the substitutions, the two hydrophobic interaction surfaces adjust to accommodate the Eth and Nle residues. Enzyme activation studies with MLCK show that Eth-CaM and Nle-CaM activate the enzyme to 90% of its maximal activity, with little changes in dissociation constant. For calcineurin only 50% activation was obtained, and the K(D) for Nle-CaM also increased 3.5-fold compared with WT-CaM. These data show that the "active site" Met residues of CaM play a distinct role in the activation of different target enzymes, in agreement with site-directed mutagenesis studies of the Met residues of CaM.

A peptide analog of the calmodulin-binding domain of myosin light chain kinase adopts an alpha-helical structure in aqueous trifluoroethanol.

A 22-residue synthetic peptide encompassing the calmodulin (CaM)-binding domain of skeletal muscle myosin light chain kinase was studied by two-dimensional NMR and CD spectroscopy. In water the peptide does not form any regular structure; however, addition of the helix-inducing solvent trifluoroethanol (TFE) causes it to form an alpha-helical structure. The proton NMR spectra of this peptide in 25% and 40% TFE were assigned by double quantum-filtered J-correlated spectroscopy, total correlation spectroscopy, and nuclear Overhauser effect correlated spectroscopy spectra. In addition, the alpha-carbon chemical shifts were obtained from (1H,13C)-heteronuclear multiple quantum coherence spectra. The presence of numerous dNN(i, i + 1), d alpha N(i, i + 3), and d alpha beta(i, i + 3) NOE crosspeaks indicates that an alpha-helix can be formed from residues 3 to 20; this is further supported by the CD data. Upfield alpha-proton and downfield alpha-carbon shifts in this region of the peptide provide further support for the formation of an alpha-helix. The helix induced by TFE appears to be similar to that formed upon binding of the peptide to CaM.

Bending of the calmodulin central helix: a theoretical study.

The crystal structure of calcium-calmodulin (CaM) reveals a protein with a typical dumbbell structure. Various spectroscopic studies have suggested that the central linker region of CaM, which is alpha-helical in the crystal structure, is flexible in solution. In particular, NMR studies have indicated the presence of a flexible backbone between residues Lys 77 and Asp 80. This flexibility is related directly to the function of the protein because it enables the N- and C-terminal domains of the protein to move toward each other and bind to the CaM-binding domain of a target protein. We have investigated the flexibility of the CaM central helix by a variety of computational techniques: molecular dynamics (MD) simulations, normal mode analysis (NMA), and essential dynamics (ED) analysis. Our MD results reproduce the experimentally determined location of the bend in a simulation of only the CaM central helix, indicating that the bending point is an intrinsic property of the alpha-helix, for which the remainder of the protein is not important. Interestingly, the modes found by the ED analysis of the MD trajectory are very similar to the lowest frequency modes from the NM analysis and to modes found by an ED analysis of different structures in a set of NMR structures. Electrostatic interactions involving residues Arg 74 and Asp 80 seem to be important for these bending motions and unfolding, which is in line with pH-dependent NMR and CD studies.

Clustered arg genes on a BamHI segment of the Escherichia coli chromosome.

BamHI cleavage of DNAs from transducing phages gamma darg13 (ppc, argECBH, bfe), gamma darg14 (ppc, argECBH) and gamma darg23 (argECBH) yields three purely gamma DNA segments (and, in one case, a fourth), as well as several Excherichia coli-DNA-containing segments. The length (in kilobases, kb) of the segments, determined by electron microscopy and ararose gel electrophoresis is 4.2, 7.5, 8.4, 6.2, 6.9, and 6.4 kb for gamma darg13; 13.0, 7.5, 4.7, 6.2, 6.9, and 6.4 kb for gamma darg 14: and 5.3, 11.0, 4.7, 6.2, 6.9, and 6.4 kb for gamma darg23. Ordering of the segments (in relation to the gamma genetic map and with the direction from left to right corresponding to the clockwise orientation of the E. coli genetic map and to each of the numerical sequences given) reveals, on 26 kb of bacterial DNA, two cleavage sites defining the 7.5-kb segment obtainable from the DNA of either gamam darg13 or gamma darg14. These and analogous findings with argEC and argCB deletion-bearing strains, together with results from heteroduplex experiments, locate argE, argC, argB, and presumably argH on the 7.5-kb segment.

NMR studies of the methionine methyl groups in calmodulin.

Calmodulin (CaM) is a ubiquitous Ca(2+)-binding protein that can regulate a wide variety of cellular events. The protein contains 9 Met out of a total of 148 amino acid residues. The binding of Ca2+ to CaM induces conformational changes and exposes two Met-rich hydrophobic surfaces which provide the main protein-protein contact areas when CaM interacts with its target enzymes. Two-dimensional (1H,13C)-heteronuclear multiple quantum coherence (HMQC) NMR spectroscopy was used to study selectively 13C-isotope labelled Met methyl groups in apo-CaM, Ca(2+)-CaM and a complex of CaM with the CaM-binding domain of skeletal muscle Myosin Light Chain Kinase (MLCK). The resonance assignment of the Met methyl groups in these three functionally different states were obtained by site-directed mutagenesis (Met-->Leu). Chemical shift changes indicate that the methyl groups of the Met residues are in different environments in apo-, calcium-, and MLCK-bound-CaM. The T1 relaxation rates of the individual Met methyl carbons in the three forms of CaM indicate that those in Ca(2+)-CaM have the highest mobility. Our results also suggest that the methyl groups of the unbranched Met sidechains in general are more flexible than those of aliphatic amino acid residues such as Leu and Ile.