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1.
Microvasc Res ; 129: 103986, 2020 05.
Article in English | MEDLINE | ID: mdl-32017943

ABSTRACT

Wet age-related macular degeneration (AMD) and diabetic retinopathy are the leading causes of blindness through increased angiogenesis. Although VEGF-neutralizing proteins provide benefit, inconsistent responses indicate a need for new therapies. We previously identified the Fibulin-7 C-terminal fragment (Fbln7-C) as an angiogenesis inhibitor in vitro. Here we show that Fbln7-C inhibits neovascularization in vivo, in both a model of wet AMD involving choroidal neovascularization (CNV) and diabetic retinopathy involving oxygen-induced ischemic retinopathy. Furthermore, a short peptide sequence from Fbln7-C is responsible for the anti-angiogenic properties of Fbln7-C. Our work suggests Fbln7-C as a therapeutic candidate for wet AMD and ischemic retinopathy.


Subject(s)
Angiogenesis Inhibitors/pharmacology , Calcium-Binding Proteins/pharmacology , Choroid/blood supply , Choroidal Neovascularization/prevention & control , Peptide Fragments/pharmacology , Retinal Neovascularization/prevention & control , Retinal Vessels/drug effects , Wet Macular Degeneration/prevention & control , Animals , Calcium-Binding Proteins/chemical synthesis , Calcium-Binding Proteins/genetics , Choroidal Neovascularization/genetics , Choroidal Neovascularization/metabolism , Choroidal Neovascularization/pathology , Disease Models, Animal , Female , Mice, Inbred C57BL , Peptide Fragments/chemical synthesis , Retinal Neovascularization/genetics , Retinal Neovascularization/metabolism , Retinal Neovascularization/pathology , Retinal Vessels/metabolism , Retinal Vessels/pathology , Wet Macular Degeneration/genetics , Wet Macular Degeneration/metabolism , Wet Macular Degeneration/pathology
3.
Biophys Chem ; 239: 16-28, 2018 08.
Article in English | MEDLINE | ID: mdl-29758467

ABSTRACT

ALG-2 dimerization was studied using Förster resonance-energy-transfer. D162C variants of ALG-2des23 were covalently modified with Alexa Fluor 488 and Alexa Fluor 647. When samples of the two labeled protein-preparations are combined, the sensitized emission from AF647 serves as a sensitive probe of dimer formation. At 25 °C, in the absence of divalent ions, the wild-type homodimer, ΔGF122 homodimer, and heterodimer display dissociation constants of 7.1, 26, and 4.5 nM, respectively. At 35 °C, subunit interaction is weaker, indicating that dimer formation is exothermic. Binding of Mg2+ in the C-terminal EF-hand (EF5) dramatically enhances ALG-2 dimer stability. Although occupation of EF5 by Ca2+ likewise has a stabilizing effect, its direct influence on dimer stability would be negligible at cytosolic Ca2+ levels. However, dimer stability is substantially increased by the Ca2+-dependent binding of ALG-2 target-peptides, suggesting that the occupation-status of the target-protein binding site is communicated to the dimer interface. Tween 20 is commonly used to improve ALG-2 solubility, the micelles ostensibly acting as target-protein surrogates. Paradoxically, however, the detergent markedly destabilizes ALG-2 dimers, particularly in the presence of Ca2+.


Subject(s)
Apoptosis Regulatory Proteins/chemistry , Apoptosis Regulatory Proteins/chemical synthesis , Calcium-Binding Proteins/chemistry , Calcium-Binding Proteins/chemical synthesis , Protein Multimerization , Calcium/chemistry , Fluorescence Resonance Energy Transfer , Humans
4.
Chem Biol Drug Des ; 81(4): 463-73, 2013 Apr.
Article in English | MEDLINE | ID: mdl-22578098

ABSTRACT

The interplay between cardiac sarcoplasmic Ca(2+)ATPase and phospholamban is a key regulating factor of contraction and relaxation in the cardiac muscle. In heart failure, aberrations in the inhibition of sarcoplasmic Ca(2+)ATPase by phospholamban are associated with anomalies in cardiac functions. In experimental heart failure models, modulation of the interaction between these two proteins has been shown to be a potential therapeutic approach. The aim of our research was to find molecules able to interfere with the inhibitory activity of phospholamban on sarcoplasmic Ca(2+)ATPase. For this purpose, a portion of phospholamban was synthesized and used as target for a phage-display peptide library screening. The cyclic peptide C-Y-W-E-L-E-W-L-P-C-A was found to bind to phospholamban (1-36) with high specificity. Its functional activity was tested in Ca(2+)uptake assays utilizing preparations from cardiac sarcoplasmic reticulum. By synthesizing and testing a series of alanine point-mutated cyclic peptides, we identified which amino acid was important for the inhibition of the phospholamban function. The structures of active and inactive alanine-mutated cyclic peptides, and of phospholamban (1-36), were determined by NMR. This structure-activity analysis allowed building a model of phospholamban -cyclic peptide complex. Thereafter, a simple pharmacophore was defined and used for the design of small molecules. Finally, examples of such molecules were synthesized and characterized as phospholamban inhibitors.


Subject(s)
Calcium-Binding Proteins/metabolism , Peptides, Cyclic/chemistry , Amino Acid Sequence , Animals , Calcium/metabolism , Calcium-Binding Proteins/antagonists & inhibitors , Calcium-Binding Proteins/chemical synthesis , Drug Design , Drug Evaluation, Preclinical , Guinea Pigs , Heart/drug effects , Humans , Models, Molecular , Myocardium/metabolism , Peptide Library , Peptides, Cyclic/chemical synthesis , Peptides, Cyclic/pharmacology , Protein Binding , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Structure-Activity Relationship
5.
J Am Chem Soc ; 134(42): 17704-13, 2012 Oct 24.
Article in English | MEDLINE | ID: mdl-22998171

ABSTRACT

We have designed a highly specific inhibitor of calpain by mimicking a natural protein-protein interaction between calpain and its endogenous inhibitor calpastatin. To enable this goal we established a new method of stabilizing an α-helix in a small peptide by screening 24 commercially available cross-linkers for successful cysteine alkylation in a model peptide sequence. The effects of cross-linking on the α-helicity of selected peptides were examined by CD and NMR spectroscopy, and revealed structurally rigid cross-linkers to be the best at stabilizing α-helices. We applied this strategy to the design of inhibitors of calpain that are based on calpastatin, an intrinsically unstable polypeptide that becomes structured upon binding to the enzyme. A two-turn α-helix that binds proximal to the active site cleft was stabilized, resulting in a potent and selective inhibitor for calpain. We further expanded the utility of this inhibitor by developing irreversible calpain family activity-based probes (ABPs), which retained the specificity of the stabilized helical inhibitor. We believe the inhibitor and ABPs will be useful for future investigation of calpains, while the cross-linking technique will enable exploration of other protein-protein interactions.


Subject(s)
Calcium-Binding Proteins/pharmacology , Calpain/antagonists & inhibitors , Cysteine Proteinase Inhibitors/pharmacology , Calcium-Binding Proteins/chemical synthesis , Calcium-Binding Proteins/chemistry , Calpain/chemistry , Calpain/metabolism , Cysteine Proteinase Inhibitors/chemical synthesis , Cysteine Proteinase Inhibitors/chemistry , Models, Molecular , Molecular Structure , Protein Binding , Protein Structure, Secondary , Structure-Activity Relationship
6.
J Am Chem Soc ; 133(45): 18406-12, 2011 Nov 16.
Article in English | MEDLINE | ID: mdl-21961692

ABSTRACT

Mice lacking the gene encoding matrix gla protein (MGP) exhibit massive mineral deposition in blood vessels and die soon after birth. We hypothesize that MGP prevents arterial calcification by adsorbing to growing hydroxyapatite (HA) crystals. To test this, we have used a combined experimental-computational approach. We synthesized peptides covering the entire sequence of human MGP, which contains three sites of serine phosphorylation and five sites of γ-carboxylation, and studied their effects on HA crystal growth using a constant-composition autotitration assay. In parallel studies, the interactions of these sequences with the {100} and {001} faces of HA were analyzed using atomistic molecular dynamics (MD) simulations. YGlapS (amino acids 1-14 of human MGP) and SK-Gla (MGP43-56) adsorbed rapidly to the {100} and {001} faces and strongly inhibited HA growth (IC(50) = 2.96 µg/mL and 4.96 µg/mL, respectively). QR-Gla (MGP29-42) adsorbed more slowly and was a moderate growth inhibitor, while the remaining three (nonpost-translationally modified) peptides had little or no effect in either analysis. Substitution of gla with glutamic acid reduced the adsorption and inhibition activities of SK-Gla and (to a lesser extent) QR-Gla but not YGlapS; substitution of phosphoserine with serine reduced the inhibitory potency of YGlapS. These studies suggest that MGP prevents arterial calcification by a direct interaction with HA crystals that involves both phosphate groups and gla residues of the protein. The strong correlation between simulated adsorption and measured growth inhibition indicates that MD provides a powerful tool to predict the effects of proteins and peptides on crystal formation.


Subject(s)
Calcinosis/prevention & control , Calcium-Binding Proteins/chemistry , Durapatite/chemistry , Extracellular Matrix Proteins/chemistry , Adsorption , Calcium-Binding Proteins/chemical synthesis , Crystallization , Extracellular Matrix Proteins/chemical synthesis , Humans , Molecular Dynamics Simulation , Matrix Gla Protein
7.
Biol Chem ; 389(1): 83-90, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18095873

ABSTRACT

The 27-mer peptide CP1B-[1-27] derived from exon 1B of calpastatin stands out among the known inhibitors for mu- and m-calpain due to its high potency and selectivity. By systematical truncation, a 20-mer peptide, CP1B-[4-23], was identified as the core sequence required to maintain the affinity/selectivity profile of CP1B-[1-27]. Starting with this peptide, the turn-like region Glu(10)(i)-Leu(11)(i+1)-Gly(12)(i+2)-Lys(13)(i+3) was investigated. Sequence alignment of subdomains 1B, 2B, 3B and 4B from different mammalians revealed that the amino acid residues in position i+1 and i+2 are almost invariably flanked by oppositely charged residues, pointing towards a turn-like conformation stabilized by salt bridge/H-bond interaction. Accordingly, using different combinations of acidic and basic residues in position i and i+3, a series of conformationally constrained variants of CP1B-[4-23] were synthesized by macrolactamization utilizing the side chain functionalities of these residues. With the combination of Glu(i)/Dab(i+3), the maximum of conformational rigidity without substantial loss in affinity/selectivity was reached. These results clearly demonstrate that the linear peptide chain corresponding to subdomain 1B reverses its direction in the region Glu(10)-Lys(13) upon binding to mu-calpain, and thereby adopts a loop-like rather than a tight turn conformation at this site.


Subject(s)
Calcium-Binding Proteins/pharmacology , Calpain/antagonists & inhibitors , Cysteine Proteinase Inhibitors/pharmacology , Amino Acid Sequence , Calcium-Binding Proteins/chemical synthesis , Calcium-Binding Proteins/chemistry , Cysteine Proteinase Inhibitors/chemical synthesis , Cysteine Proteinase Inhibitors/chemistry , Drug Design , Humans , Kinetics , Lactams/chemistry , Models, Molecular , Molecular Sequence Data , Peptides/pharmacology , Protein Conformation , Structure-Activity Relationship , Substrate Specificity
8.
Biopolymers ; 88(1): 29-35, 2007.
Article in English | MEDLINE | ID: mdl-17066471

ABSTRACT

Electron paramagnetic resonance (EPR) was used to optimize the solid-phase peptide synthesis of a membrane-bound peptide labeled with TOAC (2,2,6,6-tetramethyl-piperidine-1-oxyl-4-amino-4-carboxylic acid). The incorporation of this paramagnetic amino acid results in a nitroxide spin label coupled rigidly to the alpha-carbon, providing direct detection of peptide backbone dynamics by EPR. We applied this approach to phospholamban, which regulates cardiac calcium transport. The synthesis of this amphipathic 52-amino-acid membrane peptide including TOAC is a challenge, especially in the addition of TOAC and the next several amino acids. Therefore, EPR of synthetic intermediates, reconstituted into lipid bilayers, was used to ensure complete coupling and 9-fluorenylmethoxycarbonyl (Fmoc) deprotection. The attachment of Fmoc-TOAC-OH leads to strong immobilization of the spin label, whereas Fmoc deprotection dramatically mobilizes it, producing an EPR spectral peak that is completely resolved from that observed before deprotection. Similarly, coupling of the next amino acid (Ser) restores the spin label to strong immobilization, giving a peak that is completely resolved from that of the preceding step. For several subsequent steps, the effect of coupling and deprotection is similar but less dramatic. Thus, the sensitivity and resolution of EPR provides a quantitative monitor of completion at each of these critical steps in peptide synthesis. Mass spectrometry, circular dichroism, and Edman degradation were used in concert with EPR to verify the chemistry and characterize the secondary structure. In conclusion, the application of conventional analytical methods in combination with EPR offers an improved approach to optimize the accurate synthesis of TOAC spin-labeled membrane peptides.


Subject(s)
Calcium-Binding Proteins/chemical synthesis , Amino Acid Sequence , Biopolymers/chemistry , Calcium-Binding Proteins/chemistry , Circular Dichroism , Cyclic N-Oxides , Electron Spin Resonance Spectroscopy , Liposomes , Molecular Sequence Data , Protein Structure, Secondary , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Spin Labels , Thermodynamics
9.
Biochemistry ; 45(28): 8617-27, 2006 Jul 18.
Article in English | MEDLINE | ID: mdl-16834336

ABSTRACT

The sequence of phospholamban (PLB) is practically invariant among mammalian species. The hydrophobic transmembrane domain has 10 leucine and 8 isoleucine residues. Two roles have been proposed for the leucines; one subset stabilizes PLB oligomers, while a second subset physically interacts with SERCA. On the basis of the sequence of the PLB transmembrane domain, we chemically synthesized a series of peptides and tested their ability to regulate SERCA in reconstituted membranes. In all, eight peptides were studied: a peptide corresponding to the null-cysteine transmembrane domain of PLB (TM-Ala-PLB), two polyleucine peptides (Leu18 and Leu24), polyalanine peptides containing 4, 7, and 12 leucine residues (Leu4, Leu7, and Leu12, respectively), and a polyalanine peptide containing the 9 leucine residues present in the transmembrane domain of PLB with and without the essential Asn34 residue (Asn1Leu9 and Leu9, respectively). With the exception of Leu18, co-reconstitution of the peptides revealed effects on the apparent calcium affinity of SERCA. The TM-Ala-PLB peptide possessed approximately 70% of the inhibitory function of wild-type PLB. The remaining peptides exhibited significant inhibitory activity decreasing in the following order: Leu12, Leu9, Leu24, Leu7, and Leu4. Replacing Asn34 of PLB in the Leu9 peptide resulted in superinhibition of SERCA. On the basis of these observations, we conclude that a partial requirement for SERCA inhibition is met by a simple hydrophobic surface on a transmembrane alpha-helix. In addition, the superinhibition observed for the Asn34-containing peptide suggests that the model peptides mimic the inhibitory properties of PLB. A model is presented in which surface complementarity around key amino acid positions is enhanced in the interaction with SERCA.


Subject(s)
Calcium-Binding Proteins/chemistry , Calcium-Transporting ATPases/antagonists & inhibitors , Enzyme Inhibitors/chemistry , Peptides/chemistry , Alanine/chemistry , Amino Acid Sequence , Calcium-Binding Proteins/chemical synthesis , Calcium-Binding Proteins/pharmacology , Cell Membrane/chemistry , Cell Membrane/enzymology , Enzyme Inhibitors/pharmacology , Humans , Kinetics , Leucine/chemistry , Molecular Sequence Data , Peptides/chemical synthesis , Peptides/pharmacology , Protein Structure, Secondary , Protein Structure, Tertiary , Sarcoplasmic Reticulum Calcium-Transporting ATPases
10.
Biochemistry ; 44(44): 14688-94, 2005 Nov 08.
Article in English | MEDLINE | ID: mdl-16262268

ABSTRACT

Human psoriasin (S100A7), a member of the S100 family of calcium-binding proteins, is richly expressed in keratinocytes of patients suffering from psoriasis. To date, the exact physiological function of psoriasin abundant in many human cell types remains unclear. A recent report by Schröder and colleagues suggests that psoriasin, purified from human stratum corneum extracts, selectively kills Escherichia coli by sequestering Zn(2+) ions essential for bacterial growth, indicative of an important role in innate immune defense against microbial infection. We chemically synthesized the N-terminally acetylated psoriasin of 100 amino acid residues using solid phase peptide synthesis in combination with native chemical ligation. More than 140 mg of highly pure and correctly folded synthetic psoriasin was obtained from a single synthesis on a 0.25 mmol scale. Analysis of synthetic psoriasin by size exclusion chromatography showed that the protein forms a homodimer in solution. Circular dichroism analysis indicated that the alpha-helicity of psoriasin increases by more than 20% in the presence of CaCl(2) or ZnCl(2), suggesting a metal ion binding induced conformational change. Circular dichroism based titration further established that the synthetic protein binds two Ca(2+) and two Zn(2+) ions per dimer, in agreement with the published structural findings. Importantly, the ability of the synthetic protein to kill E. coli and the inhibition of the killing by ZnCl(2) is comparable to that of psoriasin isolated from its natural source. The robust synthetic access to large quantities of human psoriasin should facilitate studies of its biological functions as well as its mode of action.


Subject(s)
Calcium-Binding Proteins/chemical synthesis , Psoriasis/metabolism , Amino Acid Sequence , Calcium/chemistry , Calcium-Binding Proteins/chemistry , Calcium-Binding Proteins/genetics , Escherichia coli/metabolism , Humans , Molecular Sequence Data , Protein Conformation , S100 Calcium Binding Protein A7 , S100 Proteins , Zinc/chemistry
11.
Biochemistry ; 44(23): 8267-73, 2005 Jun 14.
Article in English | MEDLINE | ID: mdl-15938616

ABSTRACT

The effects of Ca(2+) binding on the dynamic properties of Ca(2+)-binding proteins are important in Ca(2+) signaling. To understand the role of Ca(2+) binding, we have successfully designed a Ca(2+)-binding site in the domain 1 of rat CD2 (denoted as Ca.CD2) with the desired structure and retained function. In this study, the backbone dynamic properties of Ca.CD2 have been investigated using (15)N spin relaxation NMR spectroscopy to reveal the effect of Ca(2+) binding on the global and local dynamic properties without the complications of multiple interactive Ca(2+) binding and global conformational change. Like rat CD2 (rCD2) and human CD2 (hCD2), residues involved in the recognition of the target molecule CD48 exhibit high flexibility. Mutations N15D and N17D that introduce the Ca(2+) ligands increase the flexibility of the neighboring residues. Ca(2+)-induced local dynamic changes occur mainly at the residues proximate to the Ca(2+)-binding pocket or the residues in loop regions. The beta-strand B of Ca.CD2 that provides two Asp for the Ca(2+) undergoes an S(2) decrease upon the Ca(2+) binding, while the DE-loop that provides one Asn and one Asp undergoes an S(2) increase. Our study suggests that Ca(2+) binding has a differential effect on the rigidity of the residues depending on their flexibility and location within the secondary structure.


Subject(s)
CD2 Antigens/chemistry , Calcium-Binding Proteins/chemical synthesis , Calcium/chemistry , Thermodynamics , Animals , Antigens, CD/metabolism , Binding Sites/genetics , CD2 Antigens/genetics , CD2 Antigens/metabolism , CD48 Antigen , Calcium/metabolism , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , EF Hand Motifs , Humans , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation , Protein Engineering , Protein Structure, Secondary/genetics , Protein Structure, Tertiary/genetics , Rats
12.
Proc Natl Acad Sci U S A ; 101(40): 14437-42, 2004 Oct 05.
Article in English | MEDLINE | ID: mdl-15448204

ABSTRACT

We have used chemical synthesis and electron paramagnetic resonance to probe the structural dynamics of phospholamban (PLB) in lipid bilayers. Derivatives of monomeric PLB were synthesized, each of which contained a single spin-labeled 2,2,6,6,-Tetramethyl-piperidine-N-oxyl-4-amino-4-carboxylic acid amino acid, with the nitroxide-containing ring covalently and rigidly attached to the alpha-carbon, providing direct insight into the conformational dynamics of the peptide backbone. 2,2,6,6,-tetramethyl-piperidine-N-oxyl-4-amino-4-carboxylic acid was attached at positions 0, 11, and 24 in the cytoplasmic domain or at position 46 in the transmembrane domain. The electron paramagnetic resonance spectrum of the transmembrane domain site (position 46) indicates a single spectral component corresponding to strong immobilization of the probe, consistent with the presence of a stable and highly ordered transmembrane helix. In contrast, each of the three cytoplasmic domain probes has two clearly resolved spectral components (conformational states), one of which indicates nearly isotropic nanosecond dynamic disorder. For the probe at position 11, an N-terminal lipid anchor shifts the equilibrium toward the restricted component, whereas Mg(2+) shifts it in the opposite direction. Relaxation enhancement, due to Ni(2+) ions chelated to lipid head-groups, provides further information about the membrane topology of PLB, allowing us to confirm and refine a structural model based on previous NMR data. We conclude that the cytoplasmic domain of PLB is in a dynamic equilibrium between an ordered conformation, which is in direct contact with the membrane surface, and a dynamically disordered form, which is detached from the membrane and poised to interact with its regulatory target.


Subject(s)
Calcium-Binding Proteins/chemistry , Lipid Bilayers/chemistry , Amino Acid Sequence , Calcium-Binding Proteins/chemical synthesis , Cyclic N-Oxides , Electron Spin Resonance Spectroscopy , In Vitro Techniques , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Protein Structure, Secondary , Protein Structure, Tertiary , Spin Labels , Thermodynamics
13.
Biochemistry ; 43(31): 9961-7, 2004 Aug 10.
Article in English | MEDLINE | ID: mdl-15287723

ABSTRACT

The alpha-lactalbumins and c-type lysozymes have virtually identical structure but exhibit very different folding behavior. All alpha-lactalbumins form a well populated molten globule state, while most of the lysozymes do not. alpha-Lactalbumin consists of two subdomains, and the alpha-subdomain is considerably more structured in the molten globule state than the beta-subdomain. Constructs derived from the alpha-subdomain of human alpha-lactalbumin containing the A, B, D, and 3(10) helices are known to form a molten globule state in the absence of the rest of the protein (Demarest, S. et al. (1999) J. Mol. Biol. 294, 213-221). Here we reported comparative studies of constructs derived from the same regions of canine and equine lysozymes. These proteins form two of the most stable molten globule states among all the lysozymes. A construct containing the A, B, D, and 3(10) helices of equine lysozyme is partially helical but is less structured than the corresponding human alpha-lactalbumin peptide. Addition of the C-helix leads to a construct that is still less structured and less stable than the alpha-lactalbumin construct. The corresponding construct from canine lysozyme is also less structured and less stable than the alpha-lactalbumin peptide. Thus, molten globule formation in human alpha-lactalbumin can be driven by the isolated alpha-subdomain, while more extensive interactions are required to generate a stable molten globule in the two lysozymes. The stability of the canine and equine lysozyme constructs is similar, indicating that the extraordinary stability of the canine lysozyme molten globule is not due to an unusually stable isolated alpha-subdomain.


Subject(s)
Calcium-Binding Proteins/metabolism , Lactalbumin/chemistry , Muramidase/metabolism , Protein Folding , Amino Acid Sequence , Animals , Calcium-Binding Proteins/chemical synthesis , Calcium-Binding Proteins/isolation & purification , Dogs , Enzyme Stability , Horses , Humans , Lactalbumin/metabolism , Molecular Sequence Data , Muramidase/chemical synthesis , Muramidase/isolation & purification , Peptide Fragments/chemical synthesis , Peptide Fragments/isolation & purification , Protein Denaturation , Protein Interaction Mapping , Protein Structure, Secondary , Protein Structure, Tertiary , Urea
14.
Biochemistry ; 43(19): 5842-52, 2004 May 18.
Article in English | MEDLINE | ID: mdl-15134458

ABSTRACT

We used EPR spectroscopy to probe directly the interaction between phospholamban (PLB) and its regulatory target, the sarcoplasmic reticulum Ca-ATPase (SERCA). Synthetic monomeric PLB was prepared with a single cytoplasmic cysteine at residue 11, which was then spin labeled. PLB was reconstituted into membranes in the presence or absence of SERCA, and spin label mobility and accessibility were measured. The spin label was quite rotationally mobile in the absence of SERCA, but became more restricted in the presence of SERCA. SERCA also decreased the dependence of spin label mobility on PLB concentration in the membrane, indicating that SERCA reduces PLB-PLB interactions. The spin label MTSSL, attached to Cys11 on PLB by a disulfide bond, was stable at position 11 in the absence of SERCA. In the presence of SERCA, the spin label was released and a covalent bond was formed between PLB and SERCA, indicating direct interaction of one or more SERCA cysteine residues with Cys11 on PLB. The accessibility of the PLB-bound spin label IPSL to paramagnetic agents, localized in different phases of the membrane, indicates that SERCA greatly reduces the level of interaction of the spin label with the membrane surface. We propose that the cytoplasmic domain of PLB associates with the lipid surface, and that association with SERCA induces a major conformational change in PLB in which the cytoplasmic domain is drawn away from the lipid surface by SERCA.


Subject(s)
Calcium-Binding Proteins/chemistry , Calcium-Transporting ATPases/metabolism , Cytoplasm/chemistry , Sarcoplasmic Reticulum/enzymology , Alanine/genetics , Animals , Calcium-Binding Proteins/chemical synthesis , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Calcium-Transporting ATPases/chemistry , Cyclic N-Oxides/metabolism , Cysteine/genetics , Cytoplasm/metabolism , Disulfides/chemistry , Disulfides/metabolism , Electron Spin Resonance Spectroscopy/methods , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Models, Chemical , Mutagenesis, Site-Directed , Phenylalanine/genetics , Protein Binding/genetics , Protein Conformation , Protein Structure, Tertiary/genetics , Rabbits , Sarcoplasmic Reticulum/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases , Spin Labels
15.
Biochemistry ; 43(9): 2596-604, 2004 Mar 09.
Article in English | MEDLINE | ID: mdl-14992597

ABSTRACT

Uncontrolled activation of calpain has been linked to tissue damage after neuronal and cardiac ischemias, traumatic spine and brain injuries, and multiple sclerosis and Alzheimer's disease. In vivo, the activity of calpain is regulated by its endogenous inhibitor calpastatin. The pathological role of calpain has been attributed to an imbalance between the activities of the protease and its inhibitor. Thus, it is possible that by reimposing functional control on the protease, the progression of calpain-mediated diseases could be slowed or eliminated. B27-WT is a 27-residue peptide (DPMSSTYIEELGKREVTIPPKYRELLA) derived from calpastatin that was previously shown to be a potent inhibitor of mu- and m-calpain. Recently, we identified two hot spots (Leu(11)-Gly(12) and Thr(17)-Ile(18)-Pro(19)) within which the amino acid residues that are key to B27-WT's bioactivity are clustered. In the work described here, the most critical residues of B27-WT, Leu(11) and Ile(18), were further probed to determine the nature of their interaction with calpain. Our results demonstrate that the side chains of both residues interact with hydrophobic pockets in calpain and that each of these interactions is indispensable for effective inhibition of calpain. Direct interactions involving the beta- and gamma-CH(2)- of the Leu(11) and Ile(18) side chains, respectively, rather than the degree of side chain branching or hydrophobicity, seemed to play a significant role in the peptide's ability to inhibit calpain. Furthermore, the minimum peptide sequence that still retained the calpain-inhibitory potency of B27-WT was found to be MSSTYIEELGKREVTIPPKYRELL.


Subject(s)
Calcium-Binding Proteins/chemistry , Calpain/antagonists & inhibitors , Cysteine Proteinase Inhibitors/chemistry , Isoleucine/chemistry , Leucine/chemistry , Peptide Fragments/chemistry , Amino Acid Sequence , Amino Acid Substitution , Animals , Autolysis , Calcium-Binding Proteins/chemical synthesis , Cysteine Proteinase Inhibitors/chemical synthesis , Humans , Hydrophobic and Hydrophilic Interactions , Molecular Sequence Data , Peptide Fragments/chemical synthesis , Peptide Library , Protein Structure, Secondary , Protein Structure, Tertiary , Structure-Activity Relationship , Swine
16.
Neurosci Res ; 47(1): 131-5, 2003 Sep.
Article in English | MEDLINE | ID: mdl-12941454

ABSTRACT

Calpain, a Ca2+-dependent neutral protease, is highly related to the pathogenesis of a variety of disorders and its inhibitors offer potential for therapeutic intervention. General calpain inhibitors, however, have the disadvantage of a lack of specificity or poor cellular permeability or oxidization under physiological conditions. Here, we developed a membrane-permeable specific calpain inhibitor by fusing calpastatin peptide (CS) and 11 poly-arginine peptides (11R). The 11R-fused CS (11R-CS) effectively penetrated across the plasma membrane of living neurons and significantly inhibited calpain activity in the cells.


Subject(s)
Calcium-Binding Proteins/pharmacokinetics , Calpain/antagonists & inhibitors , Cell Membrane Permeability/drug effects , Cysteine Proteinase Inhibitors/pharmacokinetics , Peptides/pharmacokinetics , Animals , Calcium-Binding Proteins/chemical synthesis , Calpain/metabolism , Cell Death/drug effects , Cell Death/physiology , Cell Membrane/drug effects , Cell Membrane/metabolism , Cell Membrane Permeability/physiology , Cells, Cultured , Cysteine Proteinase Inhibitors/chemical synthesis , Dose-Response Relationship, Drug , Neurons/drug effects , Neurons/metabolism , Peptides/chemical synthesis , Rats , Substrate Specificity
17.
Biopolymers ; 69(3): 283-92, 2003 Jul.
Article in English | MEDLINE | ID: mdl-12833255

ABSTRACT

We have used synthetic lipidated peptides ("peptide-amphiphiles") to study the structure and function of isolated domains of integral transmembrane proteins. We used 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis to prepare full-length phospholamban (PLB(1-52)) and its cytoplasmic (PLB(1-25)K: phospholamban residues 1-25 plus a C-terminal lysine), and transmembrane (PLB(26-52)) domains, and a 38-residue model alpha-helical sequence as a control. We created peptide-amphiphiles by linking the C-terminus of either the isolated cytoplasmic domain or the model peptide to a membrane-anchoring, lipid-like hydrocarbon tail. Circular dichroism measurements showed that the model peptide-amphiphile, either in aqueous suspension or in lipid bilayers, had a higher degree of alpha-helical secondary structure than the unlipidated model peptide. We hypothesized that the peptide-amphiphile system would allow us to study the function and structure of the PLB(1-25)K cytoplasmic domain in a native-like configuration. We compared the function (inhibition of the Ca-ATPase in reconstituted membranes) and structure (via CD) of the PLB(1-25) amphiphile to that of PLB and its isolated transmembrane and cytoplasmic domains. Our results indicate that the cytoplasmic domain PLB(1-25)K has no effect on Ca-ATPase (calcium pump) activity, even when tethered to the membrane in a manner mimicking its native configuration, and that the transmembrane domain of PLB is sufficient for inhibition of the Ca-ATPase.


Subject(s)
Calcium-Binding Proteins/chemistry , Calcium-Binding Proteins/metabolism , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Peptides/chemistry , Buffers , Calcium-Binding Proteins/chemical synthesis , Circular Dichroism , Hydrogen-Ion Concentration , Lipids/chemistry , Liposomes , Membrane Proteins/chemical synthesis , Peptides/chemical synthesis , Protein Structure, Secondary , Protein Structure, Tertiary , Structure-Activity Relationship
18.
J Am Chem Soc ; 125(20): 6165-71, 2003 May 21.
Article in English | MEDLINE | ID: mdl-12785848

ABSTRACT

Calcium ions play key roles as structural components in biomineralization and as a second messenger in signaling pathways. We have introduced a de novo designed calcium-binding site into the framework of a non-calcium-binding protein, domain 1 of CD2. The resulting protein selectively binds calcium over magnesium with calcium-binding affinity comparable to that of natural extracellular calcium-binding proteins (K(d) of 50 microM). This experiment is the first successful metalloprotein design that has a high coordination number (seven) metal-binding site constructed into a beta-sheet protein. Our results demonstrate the feasibility of designing a single calcium-binding site into a host protein, taking into account only local properties of a calcium-binding site obtained by a survey of natural calcium-binding proteins and chelators. The resulting site exhibits strong metal selectivity, suggesting that it should now be feasible to understand and manipulate signaling processes by designing novel calcium-modulated proteins with specifically desired functions and to affect their stability.


Subject(s)
CD2 Antigens/chemistry , Calcium-Binding Proteins/chemistry , Binding Sites , CD2 Antigens/metabolism , Calcium-Binding Proteins/chemical synthesis , Calcium-Binding Proteins/metabolism , Circular Dichroism , Drug Design , Fluorescence Resonance Energy Transfer , Kinetics , Metals/chemistry , Metals/metabolism , Models, Molecular , Protein Engineering/methods , Protein Structure, Tertiary , Terbium/chemistry
20.
J Mol Biol ; 313(5): 1139-48, 2001 Nov 09.
Article in English | MEDLINE | ID: mdl-11700069

ABSTRACT

The regulation of calcium levels across the membrane of the sarcoplasmic reticulum involves the complex interplay of several membrane proteins. Phospholamban is a 52 residue integral membrane protein that is involved in reversibly inhibiting the Ca(2+) pump and regulating the flow of Ca ions across the sarcoplasmic reticulum membrane during muscle contraction and relaxation. The structure of phospholamban is central to its regulatory role. Using homonuclear rotational resonance NMR methods, we show that the internuclear distances between [1-(13)C]Leu7 and [3-(13)C]Ala11 in the cytoplasmic region, between [1-(13)C]Pro21 and [3-(13)C]Ala24 in the juxtamembrane region and between [1-(13)C]Leu42 and [3-(13)C]Cys46 in the transmembrane domain of phospholamban are consistent with alpha-helical secondary structure. Additional heteronuclear rotational-echo double-resonance NMR measurements confirm that the secondary structure is helical in the region of Pro21 and that there are no large conformational changes upon phosphorylation. These results support the model of the phospholamban pentamer as a bundle of five long alpha-helices. The long extended helices provide a mechanism by which the cytoplasmic region of phospholamban interacts with residues in the cytoplasmic domain of the Ca(2+) pump.


Subject(s)
Calcium-Binding Proteins/chemistry , Calcium-Binding Proteins/metabolism , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Amino Acid Sequence , Calcium-Binding Proteins/chemical synthesis , Calcium-Transporting ATPases/metabolism , Cell Membrane/chemistry , Cell Membrane/metabolism , Dimyristoylphosphatidylcholine/metabolism , Magnetic Resonance Spectroscopy , Membrane Proteins/chemical synthesis , Models, Molecular , Molecular Sequence Data , Phosphatidylcholines/metabolism , Phosphorylation , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Rotation , Spectroscopy, Fourier Transform Infrared
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