Biomedical subjects

Karin S Akerfeldt

Publications and source records attributed to Karin S Akerfeldt.

8 recordsLinked to original sources

PubMed2005

Calbindin D28k EF-hand ligand binding and oligomerization: four high-affinity sites--three modes of action.

Calbindin D28k, a highly conserved protein with Ca2+-sensing and Ca2+-buffering capabilities, is abundant in brain and sensory neurons. This protein contains six EF-hand subdomains, four of which bind Ca2+ with high affinity. Calbindin D28k can be reconstituted from six synthetic peptides corresponding to the six EF-hands, indicating a single-domain structure with multiple interactions between the EF-hand subdomains. In this study, we have undertaken a detailed characterization of the Ca2+-binding and oligomerization properties of each individual EF-hand peptide using CD spectroscopy and analytical ultracentrifugation. Under the conditions tested, EF2 is monomeric and does not bind Ca2+, whereas EF6, which binds Ca2+ weakly, aggregates severely. We have therefore focused this study on the high-affinity binding sites, EF-hands 1, 3, 4, and 5. Our sedimentation equilibrium data show that, in the presence of Ca2+, EF-hands 1, 3, 4, and 5 all form dimers in solution in which the distribution between the monomer, dimer, and higher order oligomers differs. The processes of Ca2+ binding and oligomerization are linked to different degrees, and three main mechanisms emerge. For EF-hands 1 and 5, the dimer binds Ca2+ more strongly than the monomer and Ca2+ binding drives dimerization. For EF-hand 4, dimer formation requires only one of the monomers to be Ca2+-bound. In this case, the Ca2+ affinity is independent of dimerization. For EF-hand 3, dimerization occurs both in the absence and presence of Ca2+, while oligomerization increases in the presence of Ca2+.

Amino Acid Sequence↗

PubMed2005

Self-assembling porphyrin-modified peptides.

[structure: see text] We report the synthesis and characterization of a novel supramolecular assembly that features long-range electronic coupling between porphyrins covalently attached to a designed peptide scaffold. The resulting construct self-assembles to form extended organized aggregates in which the porphyrins engage in exciton coupling.

Hydrogen-Ion Concentration↗

PubMed2005

Deamidation and disulfide bridge formation in human calbindin D28k with effects on calcium binding.

Calbindin D(28k) (calbindin) is a cytoplasmic protein expressed in the central nervous system, which is implied in Ca(2+) homeostasis and enzyme regulation. A combination of biochemical methods and mass spectrometry has been used to identify post-translational modifications of human calbindin. The protein was studied at 37 degrees C or 50 degrees C in the presence or absence of Ca(2+). One deamidation site was identified at position 203 (Asn) under all conditions. Kinetic experiments show that deamidation of Asn 203 occurs at a rate of 0.023 h(-1) at 50 degrees C for Ca(2+)-free calbindin. Deamidation is slower for the Ca(2+)-saturated protein. The deamidation process leads to two Asp iso-forms, regular Asp and iso-Asp. The form with regular Asp 203 binds four Ca(2+) ions with high affinity and positive cooperativity, i.e., in a very similar manner to non-deamidated protein. The form with beta-aspartic acid (or iso-Asp 203) has reduced affinity for two or three sites leading to sequential Ca(2+) binding, i.e., the Ca(2+)-binding properties are significantly perturbed. The status of the cysteine residues was also assessed. Under nonreducing conditions, cysteines 94 and 100 were found both in reduced and oxidized form, in the latter case in an intramolecular disulfide bond. In contrast, cysteines 187, 219, and 257 were not involved in any disulfide bonds. Both the reduced and oxidized forms of the protein bind four Ca(2+) ions with high affinity in a parallel manner and with positive cooperativity.

Alkylation↗

PubMed2005

Redox sensitive cysteine residues in calbindin D28k are structurally and functionally important.

Human calbindin D(28k) is a Ca(2+) binding protein that has been implicated in the protection of cells against apoptosis. In this study, the structural and functional significance of the five cysteine residues present in this protein have been investigated through a series of cysteine-to-serine mutations. The mutants were studied under relevant physiological redox potentials in which conformational changes were monitored using ANS binding. Urea-induced denaturations, as monitored by intrinsic tryptophan fluorescence, were also carried out to compare their relative stability. It was shown that the two N-terminal cysteine residues undergo a redox-driven structural change consistent with disulfide bond formation. The other cysteine residues are not by themselves sufficient at inducing structural change, but they accentuate the disulfide-dependent conformational change in a redox-dependent manner. Mass spectrometry data show that the three C-terminal cysteine residues can be modified by glutathione. Furthermore, under oxidizing conditions, the data display additional species consistent with the conversion of cysteine thiols to sulfenic acids and disulfides to disulfide-S-monoxides. The biological function of calbindin D(28k) appears to be tied to the redox state of the cysteine residues. The two N-terminal cysteine residues are required for activation of myo-inositol monophosphatase, and enzyme activation is enhanced under conditions in which these residues are oxidized. Last, oxidized calbindin D(28k) binds Ca(2+) with lower affinity than does the reduced protein.

Amino Acid Sequence↗

PubMed2005

Peptides as novel smart materials.

Important challenges in biomaterials design include predicting the formation of large-scale self-assembled structures based on local atomic-level interactions and then endowing such structures with the ability to respond sensitively to environmental cues. This responsiveness is referred to as smartness. With the advent of key technological advances in imaging, peptides have recently begun to be exploited for their potential use as biomaterials, such as filaments and fibrils, hydrogels, surfactants and peptide hybrids. Peptides offer attractive features, principally because of our detailed understanding of their ability to fold into specific structures, and the rich chemistry with which their structure and function can be manipulated for environmental response.

Biocompatible Materials↗

PubMed2004

The role of electrostatic interactions in calmodulin-peptide complex formation.

The complex between calmodulin and the calmodulin-binding portion of smMLCKp has been studied. Electrostatic interactions have been anticipated to be important in this system where a strongly negative protein binds a peptide with high positive charge. Electrostatic interactions were probed by varying the pH in the range from 4 to 11 and by charge deletions in CaM and smMLCKp. The change in net charge of CaM from approximately -5 at pH 4.5 to -15 at pH 7.5 leaves the binding constant virtually unchanged. The affinity was also unaffected by mutations in CaM and charge substitutions in the peptide. The insensitivity of the binding constant to pH may seem surprising, but it is a consequence of the high charge on both protein and peptide. At low pH it is further attenuated by a charge regulation mechanism. That is, the protein releases a number of protons when binding the positively charged peptide. We speculate that the role of electrostatic interactions is to discriminate against unbound proteins rather than to increase the affinity for any particular target protein.

Amino Acid Sequence↗

PubMed2002

Synthesis and ion conductance behavior of a tetrameric alamethicin ion channel.

[structure: see text] A porphyrin-tethered construct, containing four full-length alamethicin monomers, has been synthesized and characterized. The ion conductance data of the assembly in 1 M HCl display long-lived, albeit noisy, channels that appear to be voltage-independent multiples of only one conductance state. The noise in the data is consistent with the molecular modeling studies, which indicate that the side chain of glutamine 7 of alamethicin does not fit well into the narrow pore of a parallel four-helix bundle.

Alamethicin↗

PubMed2002

Calbindin D28k exhibits properties characteristic of a Ca2+ sensor.

Calbindin D(28k) is a member of the calmodulin superfamily of Ca(2+)-binding proteins and contains six EF-hands. The protein is generally believed to function as a Ca(2+) buffer, but the studies presented in this work indicate that it may also act as a Ca(2+) sensor. The results show that Mg(2+) binds to the same sites as Ca(2+) with an association constant of approximately 1.4.10(3) m(-1) in 0.15 m KCl. The four high affinity sites in calbindin D(28k) bind Ca(2+) in a non-sequential, parallel manner. In the presence of physiological concentrations of Mg(2+), the Ca(2+) affinity is reduced by a factor of 2, and the cooperativity, which otherwise is modest, increases. Based on the binding constants determined in the presence of physiological salt concentrations, we estimate that at the Ca(2+) concentration in a resting cell calbindin D(28k) is saturated to 40-75% with Mg(2+) but to less than 9% with Ca(2+). In contrast, the protein is expected to be nearly fully saturated with Ca(2+) at the Ca(2+) level of an activated cell. A substantial conformational change is observed upon Ca(2+) binding, but only minor structural changes take place upon Mg(2+) binding. This suggests that calbindin D(28k) undergoes Ca(2+)-induced structural changes upon Ca(2+) activation of a cell. Thus, calbindin D(28k) displays several properties that would be expected for a protein involved in Ca(2+)-induced signal transmission and hence may function not only as a Ca(2+) buffer but also as a Ca(2+) sensor. Digestion patterns resulting from limited proteolysis of the protein suggest that the loop of EF-hand 2, a variant site that does not bind Ca(2+), becomes exposed upon Ca(2+) binding.

Amino Acid Sequence↗