Direct observation of peptide exchange by stable isotope enrichment.

Niu, Chien Hua; Shindo, Heisaburo; Matsuura, Shuji; Cohen, Jack S.

doi:10.1016/s0021-9258(19)85988-4

Cited by 11 publications

(1 citation statement)

References 10 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Thus formation of FTC-RNase S is a reversible reaction. In a similar experiment, Richards & Logue (1962) found that S-peptide displaces tetramethyl-S-peptide from tetramethyl-RNase S with a half-time of 60 s at pH 4.5, 22 °C, and Niu et al (1980) found that S-peptide very slowly (in hours) displaces 13C-labeled peptide residues 1-15 from combination with S-protein at 4 °C, pH 5.4. (B) Rapid Combination between FTC-S-peptide and Folded S-Protein.…”

Section: Resultsmentioning

confidence: 91%

Measurement of the refolding combination reaction between S-peptide and S-protein

Labhardt¹,

Ridge²,

Lindquist³

et al. 1983

Biochemistry

View full text Add to dashboard Cite

S-Peptide combines with S-protein during the refolding of ribonuclease S. The kinetics of combination have now been measured by a specific probe, the absorbance (492 nm) of a fluoresceinthiocarbamyl (FTC) group on lysine-7 of S-peptide. pK changes of the FTC group detect both initial combination and later, first-order, stages in folding. Combination with the slow-folding species of S-protein occurs with a half-time of 0.4 s at 50 microM, whereas complete folding takes 50 s (pH 6.8, 31 degrees C). Thus combination takes place at an early stage in folding. The second-order rate constant of the refolding combination reaction (5 X 10(4) M-1 s-1) is 100-fold smaller than that for combination with folded S-protein, which probably reflects the lower affinity of S-protein for S-peptide in the initial complex. Inhibition by S-peptide of combination between FTC-S-peptide and S-protein shows that the refolding combination reaction is specific and reversible. Both the fast-folding and slow-folding species of unfolded S-protein participate in the refolding combination reaction.

show abstract

Section: Resultsmentioning

confidence: 91%

Measurement of the refolding combination reaction between S-peptide and S-protein

Labhardt¹,

Ridge²,

Lindquist³

et al. 1983

Biochemistry

View full text Add to dashboard Cite

show abstract

Strategy for trapping intermediates in the folding of ribonuclease and for using ¹H‐NMR to determine their structures

1983

View full text Add to dashboard Cite

SynopsisThe major unfolded form of ribonuclease A is known to show well-populated structural intermediates transiently during folding a t 0"-1O0C. We describe here how the exchange reaction between D20 and peptide NH protons can be used to trap folding intermediates. The protons protected from exchange during folding can be characterized by 'H-nmr after folding is complete. The feasibility of using 'H-nmr to resolve a set of protected peptide protons is demonstrated by using a specially prepared sample of ribonuclease S in DzO in which only the peptide protons of residues 7-14 are in the 'H-form. All eight of these protected peptide protons are H-bonded. Resonance assignments made on isolated peptides containing these residues have been used to identify the protected protons. Other sets of protected protons trapped in the 'H-form can also be isolated by differential exchange, using either ribonuclease A or S. Earlier model compound studies have indicated that H-bonded folding intermediates should be unstable in water unless stabilized by additional interactions. Nevertheless, peptides derived from ribonuclease A that contain residues 3-13 do show partial helix formation in water a t low temperatures. We discuss the possibility that specific interactions between side chains can stabilize short a-helixes by nucleating the helix, and that specific interactions may also define the helix boundaries at early stages in folding.

show abstract

The design and production of semisynthetic ribonucleases with increased thermostability by incorporation of S‐peptide analogues with enhanced helical stability

1986

View full text Add to dashboard Cite

We have used protein semisynthesis to prepare four analogues of horse cytochrome c, in which the glutamic acid residue at position 66 has been removed and replaced by norvaline, glutamine, lysine and, as a methodological control, glutamic acid. This residue is quite strongly conserved in mitochondrial cytochrome c, and forms part of a cluster of acidic residues that occurs in all cytochromes c but whose function is obscure. Comparative studies of the physical and biochemical properties of the analogues have now disclosed two specific roles for Glu66 in the protein. It contributes significantly to the stabilization of the active conformation of the protein, probably by salt bridge formation, and it appears to participate in the redox-state-dependent ATP-binding site of cytochrome c. Our results also support two general views of the role of surface charged residues in cytochrome c, namely that their disposition influences both redox potential, through the electrostatic field felt at the redox centre, and the kinetics of electron transfer, through the dipole moment they generate.

show abstract

Direct observation of peptide exchange by stable isotope enrichment.

Cited by 11 publications

References 10 publications

Measurement of the refolding combination reaction between S-peptide and S-protein

Measurement of the refolding combination reaction between S-peptide and S-protein

Strategy for trapping intermediates in the folding of ribonuclease and for using ¹H‐NMR to determine their structures

The design and production of semisynthetic ribonucleases with increased thermostability by incorporation of S‐peptide analogues with enhanced helical stability

Contact Info

Product

Resources

About

Direct observation of peptide exchange by stable isotope enrichment.

Cited by 11 publications

References 10 publications

Measurement of the refolding combination reaction between S-peptide and S-protein

Measurement of the refolding combination reaction between S-peptide and S-protein

Strategy for trapping intermediates in the folding of ribonuclease and for using 1H‐NMR to determine their structures

The design and production of semisynthetic ribonucleases with increased thermostability by incorporation of S‐peptide analogues with enhanced helical stability

Contact Info

Product

Resources

About

Strategy for trapping intermediates in the folding of ribonuclease and for using ¹H‐NMR to determine their structures