Myoglobin semisynthesis: removal of the amino-terminal valine of sperm whale myoglobin and its subsequent reincorporation

DiMarchi, Richard D.; Neireiter, George W.; Garner, William H.; Gurd, Frank R. N.

doi:10.1021/bi00581a030

Cited by 10 publications

(26 citation statements)

References 33 publications

(29 reference statements)

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“…2, spectrum E), nearly identical to natural NE -acetimidylmyoglobin under the same conditions (Fig. 2, spectrum F), demonstrates the close tertiary structural similarity of the semisynthetic Mb with the native molecule (2).…”

Section: Methodsmentioning

confidence: 54%

“…The Na-NPS group was removed from fragment. 14-153 by treatment for 24 hr with 0.1 M 2-mercaptoethanol in H20, pH 7.0; the Na-Boc and 3,y-t-butyl groups were removed from fragments 6-153 The semisynthetic products were dissolved in water to a 1% concentration, and the pH was adjusted to 11.0 at 4°C (2). A stoichiometric amount of hematin dissolved in minimal 0.1 M NaOH was added dropwise and allowed to react 30 min before extensive dialysis against water.…”

Section: Methodsmentioning

confidence: 99%

“…Studies using selective, consecutive removal and substitution ofthe NH2-terminal residues ofMb by specific degradation and resynthesis (1,2) have indicated the subtle contribution of the NH2-terminal charge and the uncharged side chains ofresidues 1 and 2 to the conformation and stability of the native molecule (3). To examine further the effects of synthetic sequence alteration with respect to stability, conformation, and electrostatic and hydrophobic interactions, a method for creating internal sequence variations must be developed.…”

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confidence: 99%

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Semisynthesis of sperm whale myoglobin by fragment condensation.

Simmerman¹,

Wang²,

Horwitz³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

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Reconstruction of the sperm whale myoglobin structure was accomplished by a series of aqueous condensations of suitably protected synthetic myoglobin fragments to a large fragment prepared from the native protein. Reaction of NANT9-acetimidomyoglobin with 3-bromo-2-(2-nitrophenylsulfenyl)skatole (BNPS-skatole) yielded the fragment corresponding to residues 15-153. The covalent structure was reformed by sequential coupling of the N-hydroxysuccinimide esters of o-nitrophenylsulfenyl-L-tryptophan (residue 14) and selectively protected peptides corresponding to residues 1-5 and 6-13, which were synthesized by the solid-phase method and removed from the resin by methoxide-catalyzed methanolysis. A mixed aqueous solvent system containing methanol and N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine/trifluoroacetic acid buffer (Quadrol) solubilized the peptide and protein fragments during the condensations. Replacement of the heme moiety and immunoaffinity chromatography made possible the isolation and purification of the reconstructed native molecule. The development of this nondestructive synthetic procedure allows investigation of the structural and functional significance of individual residues by isotopic enrichment or selective amino acid substitutions.Studies using selective, consecutive removal and substitution ofthe NH2-terminal residues ofMb by specific degradation and resynthesis (1, 2) have indicated the subtle contribution of the NH2-terminal charge and the uncharged side chains ofresidues 1 and 2 to the conformation and stability of the native molecule (3). To examine further the effects of synthetic sequence alteration with respect to stability, conformation, and electrostatic and hydrophobic interactions, a method for creating internal sequence variations must be developed. A promising approach is that of semisynthesis, a technique whereby the natural product is selectively cleaved and a fragment is isolated that is suitable for rebuilding the native structure or an analog.Previous attempts at partial synthesis with Mb have utilized tryptic or CNBr fragments in which citraconyl or maleyl protecting groups were used on the e-NH2 groups (4, 5). The current strategy exploits the oxidative lability of the tryptophans at positions 7 and 14 ofthe Mb sequence by the use of3-bromo-2-(nitrophenylsulfenyl)skatole (BNPS-skatole; skatole = 3-methylindole) to effect cleavage, yielding the fragment corresponding to residues 15-153 (designated fragment 15-153) in which the E-NH2 groups have been protected previously by reaction with methyl acetimidate (1). Sequential coupling of the N-hydroxysuccinimide (HOSu) esters of o-nitrophenylsulfenyl-L-tryptophan (NPS-Trp; residue 14) and the selectively protected peptides corresponding to residues 1-5 and 6-13 (designated peptides 1-5 and 6-13) regenerated the native sequence. Isolation of reconstructed Mb capable of correctly positioning the heme moiety was accomplished by passage through successive affinity columns on which the native semisynthetic protein was se...

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Section: Methodsmentioning

confidence: 54%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Semisynthesis of sperm whale myoglobin by fragment condensation.

Simmerman¹,

Wang²,

Horwitz³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

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“…The myoglobin was maintained in the ferri form unless otherwise noted. Experimental methods for amino acid analysis, cellulose acetate electrophoresis, potentiometric hydrogen ion titrations, sequence determinations, and ultraviolet-visible spectroscopy are described elsewhere (DiMarchi et al, 1978(DiMarchi et al, , 1979. Purifications of products and intermediates by cation-exchange chromatography were typically performed with CM-Sephadex C-50 resin in phosphate buffer, ionic strength 0.01, pH 6.5, with anion-exchange chromatography performed with DEAE-Sephadex A-50 in 0.1 M Tris-HC1,' pH 9.2.…”

Section: Methodsmentioning

confidence: 99%

“…The terminal valine was cleaved as described (DiMarchi et al, 1979) by using 200 mg of dithioerythritol, 100 gL of anisole, and 10 g of trifluoroacetic acid (TFA) per gram of protein. The TFA was removed by rotary evaporation followed by vacuum.…”

Section: Methodsmentioning

confidence: 99%

Aliphatic semisynthetic variants of the amino-terminal residue of sperm whale myoglobin: enrichment with carbon-13 and determination and interpretation of terminal pK values

Busch¹,

Maskalick²,

Neireiter³

et al. 1985

Biochemistry

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The synthesis of a series of myoglobins substituted in the amino-terminal residue to provide variation in the aliphatic nature of the side chain and enrichment in 13C was accomplished by semisynthetic methods. The replacements for valine, the native first residue, included 13C-enriched glycine, alanine, valine, leucine, and isoleucine. The products were extensively characterized and found to be virtually indistinguishable by most physical methods. 13C NMR spectroscopy showed significant differences in the amino-terminal pK value, ranging from 7.72 for [Gly1]myoglobin to 7.15 for [Leu1]myoglobin. Consideration of the electrostatic effects of the charge matrix indicated a balance of interactions at this site not significantly altered by these variations in the side chain. By examination of the crystal structure, consideration of earlier work regarding the interactions of the side chain of Leu-2, and data regarding the motions of the terminal residue, it was concluded that the interaction of the side chain of the first residue with the hydrophobic cluster formed primarily by close contact of invariant residues Leu-2 and Leu-137 was the primary cause for the reduction in terminal pK values seen for the larger aliphatics. By restricting the freedom of the residue, this interaction limits the available hydration volume and consequently favors the unprotonated form of the amine. The concurrent observation of both functional elements in the series of alpha-amino-terminal residues brings out the interrelated consequences for the two categories of solvent interactions controlling structural and functional properties in a graded way.

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Max Bergmann award lecture:Macromolecular medicinal chemistry as applied to metabolic diseases

DiMarchi

Mayer

Gelfanov

et al. 2018

Journal of Peptide Science

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This review presents the scope of research presented in an October 2016 lecture pertaining to the award of the 2015 Max Bergmann Medal. The advancement in synthetic and biosynthetic chemistry as applied to the discovery of novel macromolecular drug candidates is reviewed.The evolution of the technology from the design, synthesis, and development of the first biosynthetic peptides through the emergence of peptide-based incretin agonists that function by multiple biological mechanisms is exemplified by the progression of such peptides from preclinical to clinical study. A closing section highlights recent progress made in total chemical synthesis of insulin and related peptides.

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Myoglobin semisynthesis: removal of the amino-terminal valine of sperm whale myoglobin and its subsequent reincorporation

Cited by 10 publications

References 33 publications

Semisynthesis of sperm whale myoglobin by fragment condensation.

Semisynthesis of sperm whale myoglobin by fragment condensation.

Aliphatic semisynthetic variants of the amino-terminal residue of sperm whale myoglobin: enrichment with carbon-13 and determination and interpretation of terminal pK values

Max Bergmann award lecture:Macromolecular medicinal chemistry as applied to metabolic diseases

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