Termini

The SMD simulations were based on an NMR structure of the Ig domain 127 of the cardiac titin I-band (Improta et ah, 1996). The Ig domains consist of two /9-sheets packed against each other, with each sheet containing four strands, as shown in Fig. 8b. After 127 was solvated and equilibrated, SMD simulations were carried out by fixing one terminus of the domain and applying a force to the other in the direction from the fixed terminus to the other terminus. Simulations were performed as described by Eq. (1) with V = 0.5 A/ps and if = 10 ksT/A 414 pN/A. The force-extension profile from the SMD trajectory showed a single force peak as presented in Fig. 8a. This feature agrees well with the sawtooth-shaped force profile exhibited in AFM experiments. 

The last isomerization is remarkable in that the triple bond can shift through a long carbon chain to the terminus, where it is fixed as the (kinetically) stable acetylide. The reagent is a solution of potassium diami no-propyl amide in 1,3-di-aminopropane. In some cases alkali metal amides in liquid ammonia car also bring about "contra-thermodynamic" isomerizations the reactions are successful only if the triple bond is in the 2-position. 

No 0-allylation is observed in formation of the six-membered ring compound 79 by intramolecular allylation of the /3-keto ester 78(15,57]. Intramolecular allylation is useful for lactone fonnation. On the other hand, exclusive formation of the eight-membered ring lactone 81 from 80 may be in part derived from the preference for the nucleophile to attack the less substituted terminus of the allyl system[58]. 

An amino acid sequence is ambiguous unless we know the direction m which to read It—left to right or right to left We need to know which end is the N terminus and which IS the C terminus As we saw m the preceding section carboxypeptidase catalyzed hydrolysis cleaves the C terminal ammo acid and so can be used to identify it What about the N terminus ... 

Reaction of the B chain peptide with 1 fluoro 2 4 dimtrobenzene established that phenylalanine is the N terminus... 

Sanger also determined the sequence of the A chain and identified the cysteine residues involved m disulfide bonds befween fhe A and B chains as well as m fhe disulfide linkage wifhin fhe A chain The complefe insulin sfruefure is shown m Figure 27 11 The sfruefure shown is fhaf of bovine insulin (from cattle) The A chains of human insulin and bovine insulin differ m only fwo ammo acid residues fheir B chains are identical except for the ammo acid at the C terminus... 

When Sanger s method for N terminal residue analysis was discussed you may have wondered why it was not done sequentially Simply start at the N terminus and work steadily back to the C terminus identifying one ammo acid after another The idea is fine but It just doesn t work well m practice at least with 1 fluoro 2 4 dimtrobenzene... 

Only the N terminal amide bond is broken m the Edman degradation the rest of the peptide chain remains intact It can be isolated and subjected to a second Edman procedure to determine its new N terminus We can proceed along a peptide chain by beginning with the N terminus and determining each ammo acid m order The sequence is given directly by the structure of the PTH derivative formed m each successive degradation... 

Deprotect the ammo group at the N terminus and the carboxyl group at the C terminus... 

Its N terminus with a Z group and at its C terminus as a benzyl ester can be completely deprotected m a single operation... 

Knowing how the protein chain is folded is a key ingredient m understanding the mechanism by which an enzyme catalyzes a reaction Take carboxypeptidase A for exam pie This enzyme catalyzes the hydrolysis of the peptide bond at the C terminus It is... 

An amide linkage between two a ammo acids is called a peptide bond By convention peptides are named and written beginning at the N terminus... 

Carboxypeptidase catalyzed hydrolysis can be used to identify the C terminal ammo acid The N terminus is determined by chemical means One reagent used for this purpose is Sanger s reagent 1 fluoro 2 4 dimtrobenzene (see Figure 27 9)... 

This reaction forms the basis of one method of terminal residue analysis A peptide is treated with excess hydrazine in order to cleave all the peptide linkages One of the terminal amino acids is cleaved as the free amino acid and identified all the other ammo acid residues are converted to acyl hydrazides Which amino acid is identified by hydrazmolysis the N terminus or the C terminus ... 

Critical micelle concentration (Section 19 5) Concentration above which substances such as salts of fatty acids aggre gate to form micelles in aqueous solution Crown ether (Section 16 4) A cyclic polyether that via lon-dipole attractive forces forms stable complexes with metal 10ns Such complexes along with their accompany mg anion are soluble in nonpolar solvents C terminus (Section 27 7) The amino acid at the end of a pep tide or protein chain that has its carboxyl group intact—that IS in which the carboxyl group is not part of a peptide bond Cumulated diene (Section 10 5) Diene of the type C=C=C in which a single carbon atom participates in double bonds with two others... 

N terminus (Section 27 7) The amino acid at the end of a pep tide or protein chain that has its a ammo group intact that IS the a ammo group is not part of a peptide bond... 

Two domains, t1 and t2, exist which affect the GR post-DNA binding transcription activity (37). The major (t1) transactivation domain is 185 amino acid residues ia length with a 58-tesidue a-heUcal functional cote (38). The t1 domain is located at the N terminus of the proteia the minor (t2) trans activation domain residues on the carboxy-terminal side of the DNA binding domain. 

The methods involved in the production of proteins in microbes are those of gene expression. Several plasmids for expression of proteins having affinity tails at the C- or N-terminus of the protein have been developed. These tails are usefiil in the isolation of recombinant proteins. Most of these vectors are commercially available along with the reagents that are necessary for protein purification. A majority of recombinant proteins that have been attempted have been produced in E. Coli (1). In most cases these recombinant proteins formed aggregates resulting in the formation of inclusion bodies. These inclusion bodies must be denatured and refolded to obtain active protein, and the affinity tails are usefiil in the purification of the protein. Some of the methods described herein involve identification of functional domains in proteins (see also Protein engineering). 

Human growth hormone is a single polypeptide chain of 191 amino acids (qv) having two disulfide bonds, one between Cys-53 and Cys-165, forming a large loop in the molecule, and the other between Cys-182 and Cys-189, forming a small loop near the C-terminus. The stmcture of hGH is shown in Figure 1 molecular mass is 22,125 the empirical formula is C qH 29N 262 300 7 ... 

Methionyl hGH. The first form of hGH to be produced through recombinant DNA technology was actually a derivative of hGH having one additional methionine residue at its N-terminus (11). Although technology has advanced to the stage where natural sequence hGH can easily be produced, as of this writing this derivative, referred to as methionyl hGH, is stiU produced commercially. 

Acetylated hGH A form of hGH that is acetylated at the N-terminus has been isolated and identified (15). It is not clear if acylation serves a regulatory role or is simply an artifact of the purification. 

Truncated Forms. Tmncated forms of hGH have been produced, either through the actions of enzymes or by genetic methods. 2-CAP, generated by the controlled actions of the trypsin, has the first eight residues at the N-terminus of hGH removed. Other tmncated versions of hGH have been produced by modification of the gene before expression in a suitable host. The first 13 residues have been removed to yield a derivative having distinctive biological properties (30). In this latter case the polypeptide chain is not cleaved. 

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