Exploiting the Right Side of the Ramachandran Plot:  Substitution of Glycines by<scp>d</scp>-Alanine Can Significantly Increase Protein Stability

Anil, Burcu; Song, Bowen; Tang, Yuefeng; Raleigh, Daniel P.

doi:10.1021/ja047119i

Cited by 74 publications

(90 citation statements)

References 28 publications

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“…independent of protein sequence or native-state structure (32)), the magnitude of the increase exhibited by the D-Ala B20 and D-Ala B23 analogs (⌬⌬G u 0.9 Ϯ 0.1 and 0.2 Ϯ 0.1 kcal/mole, respectively (Fig. 5A)) was less than that previously observed in (32).…”

Section: B19mentioning

confidence: 71%

“…Protein diastereomers containing corresponding D-and L-amino acid substitutions at conserved glycines exploit the unique flexibility of this achiral residue to occupy a broad range of conformations in the Ramachandran plane. Comparison of such reciprocal substitutions provides a general strategy to explore determinants of protein stability and conformational change (13,32,45).…”

Section: B25mentioning

confidence: 99%

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Chiral Mutagenesis of Insulin

Nakagawa

Hua

et al. 2006

Journal of Biological Chemistry

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Insulin contains a ␤-turn (residues B20 -B23) interposed between two receptor-binding elements, the central ␣-helix of the B chain (B9 -B19) and its C-terminal ␤-strand (B24 -B28). The turn contains conserved glycines at B20 and B23. Although insulin exhibits marked conformational variability among crystal forms, these glycines consistently maintain positive dihedral angles within a classic type-I ␤-turn. Insulin, a small globular protein secreted by pancreatic ␤ cells, plays a central role in the control of vertebrate metabolism (1). The hormone is stored in glucose-regulated secretory granules as microcrystalline arrays of Zn 2ϩ -stabilized hexamers (2) and functions as a Zn 2ϩ -free monomer. The protein contains two chains, designated A (21 residues) and B (30 residues). The structure of insulin is well characterized (3). In the T state, 3 the predominant conformation of the monomer in solution (4 -6), the A chain contains N-and C-terminal ␣-helices; the B chain contains a central ␣-helix flanked by N-and C-terminal extended segments (Fig. 1A). The product of a single-chain precursor, designated proinsulin (7), insulin contains three disulfide bridges required for its folding, stability, and function (5, 8 -10). Structure-function relationships have been extensively investigated (for review, see Ref. 11).The B chain of insulin contains two ␤-turns (boxed in Fig. 1A, see below). The first is a type-IIЈ ␤-turn comprising residues B7-B10 (black box); the second is a type-I ␤-turn comprising residues B20 -B23 (red box; ball-and-stick model in Fig. 1B). Each contains one or more conserved glycines: Gly B8 , Gly B20 , and Gly B23 (Fig. 1C). These residues exhibit positive angles ("D-glycines") and, therefore, reside on the right side of the Ramachandran plane in regions unfavorable for L-amino acids. Gly B8 , which adjoins the A7-B7 inter-chain disulfide bridge, is invariant among vertebrate insulins and highly conserved among insulin-related growth factors (black box in Fig. 1C). Mutations at B8 markedly impair the stability of insulin and the yield of chain combination (12, 13); the efficiency of recombinant expression of a single-chain precursor (mini-proinsulin) in Saccharomyces cerevisiae is likewise reduced (14,15). Whereas such unstable insulin analogs can be highly active, D-amino acid substitutions at B8 augment stability but markedly impair activity (12,13). Despite these profound stereospecific differences, the overall structures of D and L B8 analogs closely resemble native insulin (13,16).In this report we employ "chiral mutagenesis" to investigate the B20 -B23 ␤-turn. The turn sequence is notable for conserved negative and positive charges (Glu B21 and Arg B22 in eutherian mammals) flanked by glycines (Fig. 1C). Experimental design is motivated by the pattern of main-chain dihedral angles (Table 1). To investigate the interrelation of structure, activity, and stability, isomeric analogs have been synthesized in which Gly B20 or Gly B23 is substituted by D-or L-Ala; control * This work was supported i...

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Section: B19mentioning

confidence: 71%

Section: B25mentioning

confidence: 99%

Chiral Mutagenesis of Insulin

Nakagawa

Hua

et al. 2006

Journal of Biological Chemistry

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“…Such detachment motivated design of the following insulin analogs to enable interpretation in the context of the holoreceptor. (19,20). This chiral "lock" enhanced thermodynamic stability (ΔΔG u = 1.0 ± 0.2 kcal/mol; SI Appendix, Fig.…”

Section: Resultsmentioning

confidence: 99%

Protective hinge in insulin opens to enable its receptor engagement

Menting

Yang

Chan

et al. 2014

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

145

257

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Insulin provides a classical model of a globular protein, yet how the hormone changes conformation to engage its receptor has long been enigmatic. Interest has focused on the C-terminal Bchain segment, critical for protective self-assembly in β cells and receptor binding at target tissues. Insight may be obtained from truncated "microreceptors" that reconstitute the primary hormone-binding site (α-subunit domains L1 and αCT). We demonstrate that, on microreceptor binding, this segment undergoes concerted hinge-like rotation at its B20-B23 β-turn, coupling reorientation of Phe B24 to a 60°rotation of the B25-B28 β-strand away from the hormone core to lie antiparallel to the receptor's L1-β 2 sheet. Opening of this hinge enables conserved nonpolar side chains (Ile A2 , Val A3 , Val B12 , Phe B24 , and Phe B25 ) to engage the receptor. Restraining the hinge by nonstandard mutagenesis preserves native folding but blocks receptor binding, whereas its engineered opening maintains activity at the price of protein instability and nonnative aggregation. Our findings rationalize properties of clinical mutations in the insulin family and provide a previously unidentified foundation for designing therapeutic analogs. We envisage that a switch between free and receptorbound conformations of insulin evolved as a solution to conflicting structural determinants of biosynthesis and function.diabetes mellitus | signal transduction | receptor tyrosine kinase | metabolism | protein structure H ow insulin engages the insulin receptor has inspired speculation ever since the structure of the free hormone was determined by Hodgkin and colleagues in 1969 (1, 2). Over the ensuing decades, anomalies encountered in studies of analogs have suggested that the hormone undergoes a conformational change on receptor binding: in particular, that the C-terminal β-strand of the B chain (residues B24-B30) releases from the helical core to expose otherwise-buried nonpolar surfaces (the detachment model) (3-6). Interest in the B-chain β-strand was further motivated by the discovery of clinical mutations within it associated with diabetes mellitus (DM) (7). Analysis of residuespecific photo-cross-linking provided evidence that both the detached strand and underlying nonpolar surfaces engage the receptor (8).The relevant structural biology is as follows. The insulin receptor is a disulfide-linked (αβ) 2 receptor tyrosine kinase (Fig. 1A), the extracellular α-subunits together binding a single insulin molecule with high affinity (9). Involvement of the two α-subunits is asymmetric: the primary insulin-binding site (site 1*) comprises the central β-sheet (L1-β 2 ) of the first leucine-rich repeat domain (L1) of one α-subunit and the partially helical Cterminal segment (αCT) of the other α-subunit (Fig. 1A) (10). Such binding initiates conformational changes leading to transphosphorylation of the β-subunits' intracellular tyrosine kinase (TK) domains. Structures of wild-type (WT) insulin (or analogs) bound to extracellular receptor fragments were recently...

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“…We have demonstrated an alternative strategy that consists of replacing glycine residues that have positive values of ϕ with D-amino acids. Advances in peptide synthesis and native protein ligation make this a practical approach [41]. Such a substitution can enhance protein stability provided no steric clashes are introduced and provided no adverse solvation effects are introduced [42,43].…”

Section: Denatured State Interactions As a Target For Protein Designmentioning

confidence: 99%

Electrostatic interactions in the denatured state ensemble: Their effect upon protein folding and protein stability

Cho¹,

Sato²,

Horng³

et al. 2008

Archives of Biochemistry and Biophysics

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It is now recognized that the denatured state ensemble (DSE) of proteins can contain significant amounts of structure, particularly under native conditions. Well-studied examples include small units of hydrogen bonded secondary structure, particularly helices or turns as well hydrophobic clusters. Other types of interactions are less well characterized and it has often been assumed that electrostatic interactions play at most a minor role in the DSE. However, recent studies have shown that both favorable and unfavorable electrostatic interactions can be formed in the DSE. These can include surprisingly specific non-native interactions that can even persist in the transition state for protein folding. DSE electrostatic interactions can be energetically significant and their modulation either by mutation or by varying solution conditions can have a major impact upon protein stability. pH dependent stability studies have shown that electrostatic interactions can contribute up to 4 kcal mol −1 to the stability of the DSE.

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Exploiting the Right Side of the Ramachandran Plot: Substitution of Glycines byd-Alanine Can Significantly Increase Protein Stability

Cited by 74 publications

References 28 publications

Chiral Mutagenesis of Insulin

Chiral Mutagenesis of Insulin

Protective hinge in insulin opens to enable its receptor engagement

Electrostatic interactions in the denatured state ensemble: Their effect upon protein folding and protein stability

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