Role of conserved proline residues in stabilizing tryptophan synthase α subunit: Analysis by mutants with alanine or glycine

Hayashi, Seiko; Sugisaki, Yoshiko; Ogasahara, Kyoko

doi:10.1002/prot.340090203

Cited by 47 publications

(41 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This phenomenon may reflect a gain in stability in terms of resistance to degradation, as the Gly amino acids possess a wider conformational freedom than the original Pro residues. Thus, it is possible that the mutation of Pro to Gly favors the global stability of the protein (35)(36)(37) by generating a structure that is less susceptible to degradation. Similar stability increases have been detected in proTRH mutants assayed with Gly substitutions (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…ISGs, immature SGs. ⌬31-52 preproTRH Deletion of N-terminal 22 amino acids leaving intact the first 6 amino acids preceded by the signal sequence ⌬241-255 preproTRH [241][242][243][244][245][246][247][248][249][250][251][252][253][254][255] Deletion of C-terminal 15 amino acids PGL/EKA preproTRH [40][41][42] Pro-Gly-Leu replaced by Glu-Lys-Ala ⌬PGL preproTRH [40][41][42] Deletion of Pro-Gly-Leu PGL/GGG preproTRH [40][41][42] Pro-Gly-Leu replaced by Gly-Gly-Gly AVT/GGG preproTRH [34][35][36] Ala-Val-Thr replaced by Gly-Gly-Gly ⌬AVT preproTRH [34][35][36] Deletion of Ala-Val-Thr R51G/R52G preproTRH 51,52 Arg-Arg replaced by Gly-Gly PGL/AAA preproTRH 400 cells co-expressing wild type or mutant DNA and pEGFP1 were analyzed to ensure the same transfection efficiency.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Role of a Pro-sequence in the Secretory Pathway of Prothyrotropin-releasing Hormone

Romero

Çakır

Vaslet

et al. 2008

Journal of Biological Chemistry

View full text Add to dashboard Cite

The biogenesis of rat thyrotropin releasing hormone (TRH) involves the processing of its precursor (proTRH) into five biologically active TRH peptides and several non-TRH peptides where two of them had been attributed potential biological functions. This process implicates 1) proper folding of proTRH in the endoplasmic reticulum after its biosynthesis and exit to the Golgi apparatus and beyond, 2) initial processing of proTRH in the trans Golgi network and, 3) sorting of proTRH-derived peptides to the regulated secretory pathway. Previous studies have focused on elucidating the processing and sorting determinants of proTRH. However, the role of protein folding in the sorting of proTRH remains unexplored. Here we have investigated the role in the secretion of proTRH of a sequence comprising 22 amino acid residues, located at the N-terminal region of proTRH, residues 31-52. Complete deletion of these 22 amino acids dramatically compromised the biosynthesis of proTRH, manifested as a severe reduction in the steady state level of pro-TRH in the endoplasmic reticulum. This effect was largely reproduced by the deletion of only three amino acid residues, 40 PGL 42 , within the proTRH 31-52 sequence. The decreased steady state level of the mutant ⌬PGL was due to enhanced endoplasmic reticulum-associated protein degradation. However, the remnant of ⌬PGL that escaped degradation was properly processed and sorted to secretory granules. Thus, these results suggest that the N-terminal domain within the prohormone sequence does not act as "sorting signal" in late secretion; instead, it seems to play a key role determining the proper folding pathway of the precursor and, thus, its stability.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Role of a Pro-sequence in the Secretory Pathway of Prothyrotropin-releasing Hormone

Romero

Çakır

Vaslet

et al. 2008

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…microcalorimeter at the scan rate of 1.0 K/min, which is the same system described previously (8 tTo whom reprint requests should be sent at the present address.…”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

“…Two kinds of calorimeters are available for this purpose. One is a differential scanning calorimeter used to obtain thermodynamic parameters of protein unfolding (7,8), and the other is a titration calorimeter used to obtain the thermodynamic parameters of binding of a ligand by a protein (9).A mutant human lysozyme (D86/92) that has an engineered Ca2+ binding site (Ka = 5.0 x 106 M-1) was created by replacing both Gln-86 and Ala-92 with aspartic acid (10). This mutant is a suitable model for the investigation of the stabilization mechanism induced by ligand binding because the x-ray structures of the wild-type (11) and the mutant (12) lysozymes have been already solved at 1.8 A.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Entropic stabilization of a mutant human lysozyme induced by calcium binding.

Kuroki

Kawakita

Nakamura

et al. 1992

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

View full text Add to dashboard Cite

The stabilization mechanism of the mutant human lysozyme with a calcium binding site (D86/92) was investigated by using calorimetric approaches. By differential scanning calorimetry, the enthalpy change (Al) in the unfolding of holo-D86/92 was found to be 6.8 kcal/mol smaller than that of the wild-type and apo-D86/92 lysozymes at 85TC.However, the unfolding Gibbs energy change (AG) of the holo mutant was 3.3 kcal/mol greater than the apo type at 85C, indicating a significant decrease of entropy (TAS = 10.1 kcal/mol) in the presence of Ca2 . Subsequently, the Ca2+ binding process in the folded state of the mutant was analyzed by using titration isothermal calorimetry. The binding enthalpy change was estimated to be 4.5 kcal/mol, and AG was -8.1 kcal/mol at 85°C, which indicates that the binding was caused by a large increase in entropy (TAS = 12.6 kcal/mol). From these analyses, the unfolded holo mutant was determined to bind Ca2+ with a binding AG of -4.8 kcal/mol (Al = -2.6 kcal/mol, TAS = 2.2 kcal/mol) at 85°C. Therefore, the major cause of stabilization of holo-D86/92 is the decrease in entropy of the peptide chain due to Ca2+ binding to the unfolded protein.It is known that ligand binding stabilizes a protein (1-3). This kind of stabilization is considered to be very important in biological systems because the stabilization of a protein can be easily regulated by the concentration of ligand in the system (2, 4). The mechanism of the stabilization is stoichiometrically explained by the shift of the folding-unfolding equilibrium to the folded state caused by the higher affinity of ligand to the folded state (5, 6). However, the stabilization mechanism of a protein induced by ligand binding is not so clear from the thermodynamic point of view. The reactions involve not only the change of protein conformation but also the interaction of ligand with water molecules, which makes it complicated to understand the thermodynamic mechanism of the stabilization induced by ligand binding alone. Therefore, the most effective way to clarify the mechanism is the direct analysis ofboth the denaturation and the ligand binding by using calorimetric approaches in conjunction with information of the tertiary structure of a protein. Two kinds of calorimeters are available for this purpose. One is a differential scanning calorimeter used to obtain thermodynamic parameters of protein unfolding (7,8), and the other is a titration calorimeter used to obtain the thermodynamic parameters of binding of a ligand by a protein (9).A mutant human lysozyme (D86/92) that has an engineered Ca2+ binding site (Ka = 5.0 x 106 M-1) was created by replacing both Gln-86 and Ala-92 with aspartic acid (10). This mutant is a suitable model for the investigation of the stabilization mechanism induced by ligand binding because the x-ray structures of the wild-type (11) and the mutant (12) lysozymes have been already solved at 1.8 A. By using both differential scanning calorimetry (DSC) and isothermal differential titration calorimetry (DTC) it wa...

show abstract