N.M.R. studies on myelin basic protein. II. 1H N.M.R. studies of the protein and constituent peptides in aqueous solutions

1978 ◽  
Vol 31 (11) ◽  
pp. 2387 ◽  
Author(s):  
LAT Littlemore
Keyword(s):  
Chemical Shift ◽  
Myelin Basic Protein ◽  
Cathepsin D ◽  
Ring Current ◽  
Basic Protein ◽  
Tertiary Structure ◽  
Pulse Sequence ◽  
Chemical Shift Assignments ◽  
Peptide Fragment ◽  
Line Widths

1H N.M.R. spectra (270 MHz) of myelin basic protein (MBP) at pD 3.7 in D2O were obtained as a function of concentration and compared with computed spectra. Reduced line widths obtained for 0.5-mM samples and use of the convolution difference technique enabled detection of chemical shift heterogeneities for histidine, tyrosine, methionine, threonine, and isoleucine residues in the protein; this is indicative of secondary/tertiary structure. Chemical shift assignments were confirmed by the use of the Carr-Purcell A pulse sequence and selective decoupling as well as by correlation of the MBP spectrum with that of its constituent cathepsin D digest peptides. The methyl resonance from the unique methylated arginine-107 was found, and its chemical shift compared to that of NG-monomethyl-L-arginine and the methylated arginine peak in the peptide fragment, residues 90-170. The absence of ring- current effects on the methyl chemical shift precludes conformations of MBP in which the methylarginine interacts with the phenylalanine pair at residues 89 and 90.

scholarly journals Localization of sites for ionic interaction with lipid in the C-terminal third of the bovine myelin basic protein

1977 ◽  
Vol 167 (3) ◽  
pp. 583-591 ◽  
Author(s):  
A J S Jones ◽  
M G Rumsby
Keyword(s):  
Myelin Basic Protein ◽  
Basic Protein ◽  
Protein Molecule ◽  
Molar Ratio ◽  
Tryptophan Residue ◽  
Peptide Fragment ◽  
Neutral Complex ◽  
Chloroform Phase ◽  
Complex Lipids ◽  
Soluble Complexes

The myelin basic protein from bovine brain tissue was purified and the two peptides obtained by cleavage of the polypeptide chain at the single tryptophan residue were isolated. The interaction of these peptides and the intact basic protein with complex lipids was investigated by following the solubilization of lipid-protein complexes into chloroform in a biphasic solvent system. The C-terminal peptide fragment (residues 117-170) and the intact basic protein both formed chloroform-soluble complexes with acidic lipids, but not with neutral complex lipids. The N-terminal fragment (residues 1-115) did not form chloroform-soluble complexes with either acidic or neutral complex lipids. The molar ratio of lipid to protein that caused a 50% loss of protein from the upper phase to the lower chloroform phase was the same for the intact basic protein as for the smaller C-terminal peptide fragment. Phosphatidylserine and phosphatidylinositol were approximately twice as efficient as sulphatide at causing protein redistribution to the chloroform phase. The results are interpreted as indicating that the sites for ionic interactions between lipid and charged groups on the basic protein of myelin are located in the C-terminal region of the protein molecule.

Interactions of the 18.5-kDa isoform of myelin basic protein with Ca2+-calmodulin: in vitro studies using fluorescence microscopy and spectroscopy

2002 ◽  
Vol 80 (4) ◽  
pp. 395-406 ◽  
Author(s):  
David S Libich ◽  
George Harauz
Keyword(s):  
Fluorescence Microscopy ◽  
Myelin Basic Protein ◽  
Cathepsin D ◽  
Basic Protein ◽  
Dissociation Constants ◽  
Kinase Substrate ◽  
Trp Fluorescence ◽  
Binding Peptides ◽  
Carboxyl Terminal

The interactions of the 18.5-kDa isoform of myelin basic protein (MBP) with calmodulin (CaM) in vitro have been investigated using fluorescence microscopy and spectroscopy. Two forms of MBP were used: the natural bovine C1 charge isomer (bMBP/C1) and a hexahistidine-tagged recombinant murine product (rmMBP), with only minor differences in behaviour being observed. Fragments of each protein generated by digestion with cathepsin D (EC 3.4.23.5) were also evaluated. Using fluorescence microscopy, it was shown that MBP and CaM interacted in the presence of Ca2+ under a variety of conditions, including high urea and salt concentrations, indicating that the interaction was specific and not merely electrostatic in nature. Using cathepsin D digestion fragments of MBP, it was further shown that the carboxyl-terminal domain of MBP interacted with Ca2+-CaM, consistent with our theoretical prediction. Spectroscopy of the intrinsic fluorescence of the sole Trp residue of MBP showed that binding was cooperative in nature. The dissociation constants for formation of a 1:1 MBP-Ca2+-CaM complex were determined to be 2.1 ± 0.1 and 2.0 ± 0.2 μM for bMBP/C1 and rmMBP, respectively. Fluorescence spectroscopy using cathepsin D digestion fragments indicated also that the carboxyl-terminal region of each protein interacted with Ca2+-CaM, with dissociation constants of 1.8 ± 0.2 and 2.8 ± 0.9 μM for the bMBP/C1 and rmMBP fragments, respectively. These values show a roughly 1000-fold lower affinity of MBP for CaM than other CaM-binding peptides, such as myristoylated alanine-rich C-kinase substrate, that are involved in signal transduction. Key words: myelin basic protein, Ca2+-calmodulin, intrinsic Trp fluorescence, MARCKS, cathepsin D.

Deimination of Myelin Basic Protein. 1. Effect of Deimination of Arginyl Residues of Myelin Basic Protein on Its Structure and Susceptibility to Digestion by Cathepsin D†

Biochemistry ◽  
2000 ◽  
Vol 39 (18) ◽  
pp. 5374-5381 ◽  
Author(s):  
Laura B. Pritzker ◽  
Shashikant Joshi ◽  
Jessica J. Gowan ◽  
George Harauz ◽  
Mario A. Moscarello
Keyword(s):  
Myelin Basic Protein ◽  
Cathepsin D ◽  
Basic Protein ◽  
Arginyl Residues

Acidic lipids enhance cathepsin D cleavage of the myelin basic protein

1986 ◽  
Vol 15 (2) ◽  
pp. 137-145 ◽  
Author(s):  
K. R. Williams ◽  
N. D. Williams ◽  
W. Konigsberg ◽  
R. K. Yu
Keyword(s):  
Myelin Basic Protein ◽  
Cathepsin D ◽  
Basic Protein ◽  
Acidic Lipids

N.M.R. studies of myelin basic protein. V. 1H N.M.R. of the peptides encephalitogenic in guinea pig

1981 ◽  
Vol 34 (7) ◽  
pp. 1373 ◽  
Author(s):  
SA Margetson ◽  
WJ Moore ◽  
WA Gibbons
Keyword(s):  
Myelin Basic Protein ◽  
Ring Current ◽  
Basic Protein ◽  
Chemical Shifts ◽  
C Terminus ◽  
Proton Resonances ◽  
Nuclear Overhauser ◽  
Nuclear Overhauser Effects ◽  
Peptide Data ◽  
Made In

The 1H n.m.r. spectra of the following peptides have been investigated: (1) the encephalitogenic H-Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Lys-OH comprising residues 114-122 of bovine myelin basic protein, (2) the corresponding -Arg-OH peptide of human basic protein, (3) the inactive peptide in which D-Ala5 replaces L-Ala5. Measurements were made in D2O solutions at 270 and 600 MHz over a range of temperatures, concentrations and pH. All the proton resonances have been assigned by comparisons with other peptide data, titration shifts, selective decoupling and nuclear Overhauser effects, and data on the (α,α- 2H2)Gly7 nonapeptide. Ring current shifts and their temperature dependence indicated that there is preferential stacking of the Phe and Trp rings, and also interactions between these rings and the Gln and Lys residues near the C-terminus of the peptide. These data suggest a reverse turn at the Gly4-Ala5 residues, a conformation that would be consistent with results from energy calculations and biological activity. The n.m.r. spectra of the L-Ala and D-Ala peptides differed in the temperature coefficients of certain chemical shifts, including particularly those subject to ring current effects. Data in dimethyl sulfoxide were limited by effects of aggregation, but definite conformation differences compared to aqueous solutions were indicated.

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