Biomedical subjects

H J Vogel

Publications and source records attributed to H J Vogel.

At least 19 recordsLinked to original sources

PubMed2005

Lactoferricin: a lactoferrin-derived peptide with antimicrobial, antiviral, antitumor and immunological properties.

The peptide lactoferricin (Lfcin) can be released from the multifunctional protein lactoferrin (LF) through proteolysis by pepsin under acidic conditions, a reaction that occurs naturally in the stomach. Lfcin encompasses a large portion of the functional domain of the intact protein, and in many cases it not only retains the activities of LF but is more active. Lfcin possesses strong antimicrobial and weak antiviral activities, and it also has potent antitumor and immunological properties. This review covers the current state of research in this field, focusing on the many beneficial activities of this peptide. Throughout we will discuss the breadth of Lfcin activity as well as the mechanism of action. Many recent studies have drawn attention to the fact that the main site of action for the peptide may be intracellular. In addition the results of structural and dynamic studies of Lfcin are presented, and the relationship between structure and activity is explored.

Adjuvants, Immunologic↗

PubMed2005

Interactions of the antimicrobial peptide Ac-FRWWHR-NH(2) with model membrane systems and bacterial cells.

The acetylated and amidated hexapeptide FRWWHR (combi-2), previously identified by combinatorial chemistry methods, shows strong antimicrobial activity. The binding of the peptide to 1-palmitoyl-2-oleoyl-sn-glycero-3-[(phospho-rac-(1-glycerol)] (POPG) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) vesicles was studied using fluorescence spectroscopy and isothermal titration calorimetry (ITC). Differential scanning calorimetry (DSC) with dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) multilamellar vesicles was performed to determine changes in the lipid phase behaviour upon binding the peptide. Two-dimensional proton nuclear magnetic resonance (NMR) spectroscopy, to solve the bound peptide structure, was performed in the presence of dodecylphosphatidylcholine (DPC) and sodium dodecyl sulphate (SDS) micelles. The fluorescence, ITC and DSC studies indicate that the peptide interacts preferentially with lipid vesicles containing negatively charged head groups. Conformational information determined using NMR indicate that the combi-2 peptide adopts a coiled amphipathic conformation when bound to SDS and DPC micelles. Leakage assays indicate that the peptide is not very efficient at causing leakage from calcein-filled large unilamellar vesicles comprised of POPG/POPC (1 : 1). The rapid passage of either the fluorescent-tagged peptides combi-2 or the previously studied peptide Ac-RRWWRF-NH(2) (combi-1) into Escherichia coli and Staphylococcus aureus suggests that instead of membrane disruption, the main bactericidal site of action of these peptides might be located inside bacteria.

Antimicrobial Cationic Peptides↗

PubMed2003

The structure of the antimicrobial peptide Ac-RRWWRF-NH2 bound to micelles and its interactions with phospholipid bilayers.

The hexapeptide Ac-RRWWRF-NH2 has earlier been identified as a potent antimicrobial peptide by screening synthetic combinatorial hexapeptide libraries. In this study, it was found that this peptide had a large influence on the thermotropic phase behavior of model membranes containing the negatively charged headgroup phosphatidylglycerol, a major component of bacterial membranes. In contrast, differential scanning calorimetry showed that it had little effect on model membranes containing the zwitterionic phosphatidylcholine headgroup, the main component of erythrocyte membranes. This behavior is consistent with its biological activity and with its affinity to these membranes as determined by titration calorimetry, implying that peptide-lipid interactions play an important role in this process. The structure of this peptide bound to membrane-mimetic sodium dodecyl sulfate (SDS) and dodecylphosphocholine micelles has been determined using conventional two-dimensional nuclear magnetic resonance methods. It forms a marked amphipathic structure in SDS with its hydrophobic residues on one side of the structure and with the positively charged residues on the other side. This amphipathic structure may allow this peptide to penetrate deeper into the interfacial region of negatively charged membranes, leading to local membrane destabilization. Knowledge about the importance of electrostatic interactions of Arg and the role of Trp residues as a membrane interface anchor will provide insight into the future design of potent antimicrobial peptidomimetics.

Amino Acid Sequence↗

PubMed2002

Characterization of tetrachlorohydroquinone reductive dehalogenase from Sphingomonas sp. UG30.

Tetrachlorohydroquinone reductive dehalogenase (PcpC) is the second of three enzymes that catalyze the initial degradation of pentachlorophenol in Sphingomonas sp. UG30 and several other bacterial strains. The UG30 PcpC shares a high degree (94%) of primary sequence identity with the well-studied PcpC from Sphingobium chlorophenolicum ATCC 39723. Significant differences, however, were observed between the two PcpC enzymes in some of their functional and kinetic properties. The temperature optimum of the UG30 PcpC is 10 degrees C higher and the pH optimum is approximately 2 units higher than the S. chlorophenolicum PcpC. In addition, the S. chlorophenolicum PcpC is subject to inhibition by the substrate tetrachlorohydroquinone (TCHQ), and this has necessitated the use of a mutant enzyme, which was not inhibited by TCHQ, for kinetic studies. In contrast, the UG30 PcpC was not inhibited by TCHQ and this may allow detailed kinetic and mechanistic studies using the wild-type enzyme.

Amino Acid Sequence↗

PubMed2001

The membrane-proximal tryptophan-rich region of the HIV glycoprotein, gp41, forms a well-defined helix in dodecylphosphocholine micelles.

The membrane-proximal tryptophan-rich region of the HIV transmembrane glycoprotein, gp41, plays an important role in the membrane fusion reaction. Using NMR spectroscopy, we have studied the tertiary structure of a synthetic 19-residue amidated peptide (NH2-KWASLWNWFNITNWLWYIK-CONH2) corresponding to this region in membrane-mimetic environments. Initial experiments in sodium dodecyl sulfate/H2O micelles and trifluoroethanol gave poor results, because of low solubility. However, in dodecylphosphocholine micelles, we obtained excellent 500 and 800 MHz NMR spectra, suggesting that the peptide has a preference for a zwitterionic membrane-like environment. The final NMR structures demonstrated a well-defined helical peptide with a backbone rmsd of 0.47 +/- 0.18 A. Four of the five tryptophan residues, as well as the tyrosine residue, formed a "collar" of aromatic residues along the axial length of the helix. By analogy to related tryptophan-rich antimicrobial peptides, the structure indicates that the aromatic residues of the HIV peptide are positioned within the membrane-water interface of a phospholipid bilayer. This is confirmed by the observation of direct NOEs between the aromatic residues of the peptide to the headgroup and interfacial protons of prototonated dodecylphosphocholine. The bulk of the polar residues are positioned on one face of this structure, with the hydrophobic phenylalanine side chain on the opposing face, forming an amphipathic structure. This work shows that the Trp-rich membrane-proximal region of HIV and related viruses can bind to the surfaces of zwitterioninc membranes in a "Velcro-like" manner.

Amino Acid Sequence↗

PubMed2001

Energetics of target peptide binding by calmodulin reveals different modes of binding.

Thermodynamic parameters of interactions of calcium-saturated calmodulin (Ca(2+)-CaM) with melittin, C-terminal fragment of melittin, or peptides derived from the CaM binding regions of constitutive (cerebellar) nitric-oxide synthase, cyclic nucleotide phosphodiesterase, calmodulin-dependent protein kinase I, and caldesmon (CaD-A, CaD-A*) have been measured using isothermal titration calorimetry. The peptides could be separated into two groups according to the change in heat capacity upon complex formation, DeltaC(p). The calmodulin-dependent protein kinase I, constitutive (cerebellar) nitric-oxide synthase, and melittin peptides have DeltaC(p) values clustered around -3.2 kJ.mol(-1).K(-1), consistent with the formation of a globular CaM-peptide complex in the canonical fashion. In contrast, phosphodiesterase, the C-terminal fragment of melittin, CaD-A, and CaD-A* have DeltaC(p) values clustered around -1.6 kJ.mol(-1).K(-1), indicative of interactions between the peptide and mostly one lobe of CaM, probably the C-terminal lobe. It is also shown that the interactions for different peptides with Ca(2+)-CaM can be either enthalpically or entropically driven. The difference in the energetics of peptide/Ca(2+)-CaM complex formation appears to be due to the coupling of peptide/Ca(2+)-CaM complex formation to the coil-helix transition of the peptide. The binding of a helical peptide to Ca(2+)-CaM is dominated by favorable entropic effects, which are probably mostly due to hydrophobic interactions between nonpolar groups of the peptide and Ca(2+)-CaM. Applications of these findings to the design of potential CaM inhibitors are discussed.

Amino Acid Sequence↗

PubMed2001

Calmodulin binding properties of peptide analogues and fragments of the calmodulin-binding domain of simian immunodeficiency virus transmembrane glycoprotein 41.

The calcium-regulatory protein calmodulin (CaM) can bind with high affinity to a region in the cytoplasmic C-terminal tail of glycoprotein 41 of simian immunodeficiency virus (SIV). The amino acid sequence of this region is (1)DLWETLRRGGRW(13)ILAIPRRIRQGLELT(28)L. In this work, we have used near- and far-uv CD, and fluorescence spectroscopy, to study the orientation of this peptide with respect to CaM. We have also studied biosynthetically carbon-13 methyl-Met calmodulin by (1)H, (13)C heteronuclear multiple quantum coherence NMR spectroscopy. Two Trp-substituted peptides, SIV-W3F and SIV-W12F, were utilized in addition to the intact SIV peptide. Two half-peptides, SIV-N (residues 1-13) and SIV-C (residues 13-28) were also synthesized and studied. The spectroscopic results obtained with the SIV-W3F and SIV-W12F peptides were generally consistent with those obtained for the native SIV peptide. Like the native peptide, these two analogues bind with an alpha-helical structure as shown by CD spectroscopy. Fluorescence intermolecular quenching studies suggested binding of Trp3 to the C-lobe of CaM. Our NMR results show that SIV-N can bind to both lobes of calcium-CaM, and that it strongly favors binding to the C-terminal hydrophobic region of CaM. The SIV-C peptide binds with relatively low affinity to both halves of the protein. These data reveal that the intact SIV peptide binds with its N-terminal region to the carboxy-terminal region of CaM, and this interaction initiates the binding of the peptide. This orientation is similar to that of most other CaM-binding domains.

Amino Acid Sequence↗

PubMed2001

[Mesenchymal chondrosarcomas of the facial skull].

A 13-year-old girl suffered from a mesenchymal chondrosarcoma of the left maxilla. The therapeutic options and the prognosis for this disease are described with respect to the currently known 72 cases in the literature. Mesenchymal chondrosarcomas are rare tumors of the bone and soft tissue. The first clinical symptom is a painless swelling of the facial skull. They occur largely in the 2nd and 3rd decades of life, preferentially in males. Radiological criteria for the identification of this type of tumor include focal ossification areas which are accompanied by non-calcified regions. Complete surgical removal of the tumor is the therapy of choice. Pre- and postoperative chemotherapy can have a beneficial effect. The final outcome of the disease is difficult to evaluate since late complications (e.g., reoccurrence and/or metastases) appear even after 20 years and only a small number of cases have been reported. At present, the 5-year survival rate is reported to be 54-82% and the 10-year rate 28-56%.

Adolescent↗

PubMed2001

Molecular dynamics study of peptide-bilayer adsorption.

Two 6-ns simulations of the somatostatin analog sandostatin and a 1-palmityl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer are presented. In the first simulation, the peptide was placed in a region of bulk water density and allowed to spontaneously move toward and bind to the bilayer surface. An attractive force between the peptide and bilayer drove the binding process, which was opposed by a significant frictional force caused by the solvent (water). During the approach of the peptide toward the bilayer the area of the interacting surface between the species was inversely proportional to the distance between them, supporting the application of such a relationship in continuum calculations of peptide-bilayer binding free energies. In the second simulation, the N-terminus of the surface-bound peptide was deprotonated. Consistent with experiment, this strengthened interactions between the peptide and the bilayer. Details of both peptide-bilayer complexes, including the orientation, percent buried surface area, and orientation of the lipid headgroups are in good agreement with those obtained from experiment. The location of the different side chains in the bilayer is in direct correlation with an interfacial hydrophobicity scale developed using model peptides. The aromatic side chains of the Phe and Trp residues all lie flat with respect to the bilayer surface in both complexes. Changes in lipid and water ordering due to peptide binding suggest a possible domination of lipophobic over hydrophobic effects, as proposed by other workers. Where appropriate, peptide and lipid properties in the bound states are compared with separate simulations of sandostatin and the bilayer in water, respectively, so as to monitor the response of the system to the binding process.

Adsorption↗

PubMed2001

Phosphorus-31 nuclear magnetic resonance study of the effect of pentachlorophenol (PCP) on the physiologies of PCP-degrading microorganisms.

Free and agarose-encapsulated pentachlorophenol (PCP)-degrading Sphingomonas sp. isolates UG25 and UG30 were compared to Sphingomonas chlorophenolica ATCC 39723 with respect to the ability to degrade PCP. Pretreatment of the UG25 and UG30 strains with 50 microg of PCP per ml enabled the cells to subsequently degrade higher levels of this environmental pollutant. Similar treatment of ATCC 39723 cells had no effect on the level of PCP degraded by this strain. Phosphorus-31 nuclear magnetic resonance spectra of agarose-immobilized strains UG25 and UG30 grown in the absence of PCP showed that there was marked deenergization of the cells upon exposure to a nonlethal concentration of PCP (120 microg/ml). For example, no transmembrane pH gradient was observed, and the ATP levels were lower than the levels obtained in the absence of PCP. The transmembrane pH gradient and ATP levels were restored once the immobilized cells had almost completely degraded the PCP in the perfusion medium. PCP-pretreated cells, on the other hand, maintained their transmembrane pH gradient and ATP levels even in the presence of high levels of PCP. The ability of PCP-pretreated strain UG25 and UG30 cells to remain energized in the presence of PCP was shown to correlate with an altered membrane phospholipid profile; these cells had a higher concentration of cardiolipin than cells cultured in the absence of PCP. Strain ATCC 39723, which did not degrade higher levels of PCP after PCP pretreatment, did not show this response.

Biodegradation, Environmental↗

PubMed2001

Structural biology of bacterial iron uptake systems.

Numerous bacterial proteins are involved in microbial iron uptake and transport and considerable variation has been found in the uptake schemes used by different bacterial species. However, whether extracting iron from host proteins such as transferrin, lactoferrin or hemoglobin or importing low molecular weight iron-chelating compounds such as heme, citrate or siderophores, Gram-negative pathogenic bacteria typically employ a specific outer membrane receptor, a periplasmic binding protein and two inner membrane associated proteins: a transporter coupled with an ATP-hydrolyzing protein. Often, studies have shown that proteins with similar function but little amino acid sequence homology are structurally related. Elucidation of the structures of the Escherichia coli outer membrane siderophore transport proteins FepA and FhuA have provided the first insights into the conformational changes required for ligand transport through the bacterial outer membrane. The variations between the structures of the prototypical periplasmic ferric binding protein FbpA from Neisseria and Haemophilus influenzae and the unusual E coli periplasmic siderophore binding protein FhuD reveal that the different periplasmic ligand binding proteins exercise distinct mechanisms for ligand binding and release. The structure of the hemophore HasA from Serratia marcescens shows how heme may be extracted and utilized by the bacteria. Other biochemical evidence also shows that the proteins that provide energy for iron transport at the outer membrane, such as the TonB-ExbB-ExbD system, are structurally very similar across bacterial species. Likewise, the iron-sensitive gene regulatory protein Fur is found in most bacteria. To date, no structural information is available for Fur, but the structure for the related protein DxtR has been determined. Together, these three-dimensional structures complement our knowledge of iron transport systems from other pathogenic bacteria, including Pseudomonas aeruginosa, which has a number of homologous iron uptake proteins. More importantly, the current structures for iron transport proteins provide rational starting points for design of novel antimicrobial agents.

Bacteria↗

PubMed2000

pK(a) calculations of calbindin D(9k): effects of Ca(2+) binding, protein dielectric constant, and ionic strength.

Calbindin is a small (75 residues) helix-loop-helix ("EF-hand") calcium-binding protein belonging to the calmodulin superfamily. It binds two Ca(2+) ions. Continuum electrostatics in combination with the boundary element method was employed for the calculation of the acid-dissociation constants K(a) (pK(a) = -log K(a)) values of all titratable residues in the protein. The objectives were to determine quantitatively the effects of divalent ion binding and small ion-induced structural changes on predicted pK(a)'s. Computations were carried out for the apo and holo form of calbindin, for which both X-ray and NMR structures were available. Comparison was made with several sets of experimental pK(a) values determined by NMR spectroscopy. Different choices of the dielectric constant (ranging from 4 to 78.5) for calbindin and variations in ionic strength (from 0 to 0.3 M) were investigated in a systematic fashion. Removal of the two bound Ca(2+) ions increases the pK(a) values of all residues if no conformational changes were allowed. If conformational differences between the apo and holo were accounted for, shifts in either direction were observed. Titrating groups that are directly involved in Ca(2+) binding (Asp and Glu) required a dielectric constant of 78.5 for the holo structure to obtain a reasonable estimate of their pK(a)'s. For the apo structure, passable values for the pK(a)'s of these ligating groups could be determined if the structure was allowed to relax upon ion removal.

Binding Sites↗

PubMed2000

Theoretical calculation of pKa reveals an important role of Arg205 in the activity and stability of Streptomyces sp. N174 chitosanase.

Based on the crystal structure of chitosanase from Streptomyces sp. N174, we have calculated theoretical pK(a) values of the ionizable groups of this protein using a combination of the boundary element method and continuum electrostatics. The pK(a) value obtained for Arg(205), which is located in the catalytic cleft, was abnormally high (>20.0), indicating that the guanidyl group may interact strongly with nearby charges. Chitosanases possessing mutations in this position (R205A, R205H, and R205Y), produced by Streptomyces lividans expression system, were found to have less than 0.3% of the activity of the wild type enzyme and to possess thermal stabilities 4-5 kcal/mol lower than that of the wild type protein. In the crystal structure, the Arg(205) side chain is in close proximity to the Asp(145) side chain (theoretical pK(a), -1.6), which is in turn close to the Arg(190) side chain (theoretical pK(a), 17.7). These theoretical pK(a) values are abnormal, suggesting that both of these residues may participate in the Arg(205) interaction network. Activity and stability experiments using Asp(145)- and Arg(190)-mutated chitosanases (D145A and R190A) provide experimental data supporting the hypothesis derived from the theoretical pK(a) data and prompt the conclusion that Arg(205) forms a strong interaction network with Asp(145) and Arg(190) that stabilizes the catalytic cleft.

Amino Acid Sequence↗

PubMed2000

Spectroscopic characterization of the interaction between calmodulin-dependent protein kinase I and calmodulin.

Calmodulin-dependent protein kinase I (CaM kinase I) is a member of the expanding class of protein kinases that are regulated by calmodulin (CaM). Its putative CaM-binding region is believed to occur within a 22-residue sequence (amino acids 299-320). This sequence was chemically synthesized and utilized for CaM interaction studies. Gel band shift assays and densitometry experiments with intact CaM kinase I and the CaM-binding domain peptide (CaMKIp) reveal that they bind in an analogous manner, giving rise to 1:1 complexes. Fluorescence analysis using dansyl-CaM showed that conformational changes in CaM on binding CaM kinase I or CaMKIp were nearly identical, suggesting that the peptide mimicked the CaM-binding ability of the intact protein. In the presence of CaM, the peptide displays an enhancement of its unique Trp fluorescence as well as a marked blue shift of the emission maximum, reflecting a transfer to a more rigid, less polar environment. Quenching studies, using acrylamide, confirmed that the Trp in the peptide on binding CaM is no longer freely exposed to solvent as is the case for the free peptide. Studies with a series of Met mutants of CaM showed that the Trp-containing N-terminal end of CaMKIp was bound to the C-terminal lobe of CaM. Near-UV CD spectra also indicate that the Trp of the peptide and Phe residues of the protein are involved in the binding. These results show that the CaM-binding domain of CaM kinase I binds to CaM in a manner analogous to that of myosin light chain kinase.

Acrylamide↗

PubMed2000

Comparative modeling studies of the calmodulin-like domain of calcium-dependent protein kinase from soybean.

Calmodulin-like domain protein kinases (CDPKs) represent a new class of calcium-dependent protein-phosphorylating enzymes that are not activated by calmodulin or phospholipid compounds. They have been found exclusively in plant and protozoal tissues. CDPKs are typified by four distinct domains: an N-terminal leader sequence, a protein kinase (PK) domain, a calmodulin-like domain (CLD), and a junction domain (JD) between the PK domain and CLD. Structural characterization of the CLD of CDPKalpha from soybean was undertaken based on the amino acid sequence homology of CLD to the structurally well-characterized calmodulin (CaM) family of structures. Tertiary models of apo-CLD, Ca(2+)-CLD complex, and intermolecularly bound Ca(2+)-CLD-JD complexes were obtained via automated and non-automated homology building methods. The resulting structures were compared and validated based on energy differences, phi-psi angle distribution, solvent accessibility, and hydrophobic potential. Circular dichroism, one-dimensional, and two-dimensional nuclear magnetic resonance spectroscopy studies of the CLD and peptides encompassing the JD provide experimental support to the models. The results suggest that there is a possible interaction between the CLD and JD domain similar to that of the CaM/calmodulin-dependent protein kinase II system. At low Ca(2+) levels, the JD may act as an autoinhibitory domain for kinase activity, and during calcium activation an intramolecular CLD-JD complex may form, relieving inhibition of the PK domain. Interactions between the JD and the C terminus of the CLD appear to be particularly important. The outcome of this study supports an intramolecular binding model for calcium activation of CDPK, although not exclusively.

Amino Acid Sequence↗

PubMed2000

Mapping the interface between calmodulin and MARCKS-related protein by fluorescence spectroscopy.

MARCKS-related protein (MRP) is a myristoylated protein kinase C substrate that binds calmodulin (CaM) with nanomolar affinity. To obtain structural information on this protein, we have engineered 10 tryptophan residues between positions 89 and 104 in the effector domain, a 24-residue-long amphipathic segment that mediates binding of MRP to CaM. We show that the effector domain is in a polar environment in free MRP, suggesting exposure to water, in agreement with a rod-shaped structure of the protein. The effector domain participates in the binding of MRP to CaM, as judged by the dramatic changes observed in the fluorescent properties of the mutants on complex formation. Intermolecular quenching of the fluorescence emission of the tryptophan residues in MRP by selenomethionine residues engineered in CaM reveals that the N-terminal side of the effector domain contacts the C-terminal domain of CaM, whereas the C-terminal side of the effector domain contacts the N-terminal domain of CaM. Finally, a comparison of the fluorescent properties of the myristoylated and unmyristoylated forms of a construct in which a tryptophan residue was introduced at position 4 close to the myristoylated N terminus of MRP suggests that the lipid moiety is also involved in the interaction of MRP with CaM.

Amino Acid Sequence↗

PubMed2000

The structure of the ferric siderophore binding protein FhuD complexed with gallichrome.

Siderophore binding proteins play a key role in the uptake of iron in many gram-positive and gram-negative bacteria. FhuD is a soluble periplasmic binding protein that transports ferrichrome and other hydroxamate siderophores. The crystal structure of FhuD from Escherichia coli in complex with the ferrichrome homolog gallichrome has been determined at 1.9 ¿ resolution, the first structure of a periplasmic binding protein involved in the uptake of siderophores. Gallichrome is held in a shallow pocket lined with aromatic groups; Arg and Tyr side chains interact directly with the hydroxamate moieties of the siderophore. FhuD possesses a novel fold, suggesting that its mechanisms of ligand binding and release are different from other structurally characterized periplasmic ligand binding proteins.

Bacterial Outer Membrane Proteins↗

PubMed2000

Tryptophan fluorescence of calmodulin binding domain peptides interacting with calmodulin containing unnatural methionine analogues.

The interactions between the abundant methionine residues of the calcium regulatory protein calmodulin (CaM) and several of its binding targets were probed using fluorescence spectroscopy. Tryptophan steady-state fluorescence from peptides encompassing the CaM-binding domains of the target proteins myosin light chain kinase (MLCK), cyclic nucleotide phosphodiesterase (PDE) and caldesmon site A and B (CaD A, CaD B), and the model peptide melittin showed Ca(2+)-dependent blue-shifts in their maximum emission wavelength when complexed with wild-type CaM. Blue-shifts were also observed for complexes in which the CaM methionine residues were replaced by selenomethionine, norleucine and ethionine, and when a quadruple methionine to leucine C-terminal mutant of CaM was studied. Quenching of the tryptophan fluorescence intensity was observed with selenomethionine, but not with norleucine or ethionine substituted protein. Fluorescence quenching studies with added potassium iodide (KI) demonstrate that the non-native proteins limit the solvent accessibility of the Trp in the MLCK peptide to levels close to that of the wild-type CaM-MLCK interaction. Our results show that the methionine residues from CaM are highly sensitive to the target peptide in question, confirming the importance of their role in binding interactions. In addition, we provide evidence that the nature of binding in the CaM-CaD B complex is unique compared with the other complexes studied, as the Trp residue of this peptide remains partially solvent exposed upon binding to CaM.

3',5'-Cyclic-AMP Phosphodiesterases↗