Biomedical subjects

Matthew P Hinderaker

Publications and source records attributed to Matthew P Hinderaker.

4 recordsLinked to original sources

PubMed2003

Protein prosthesis: a nonnatural residue accelerates folding and increases stability.

Nonnatural residues can endow proteins with desirable properties. Here, replacing a proline residue that has a cis peptide bond in native ribonuclease A with 5,5-dimethyl-l-proline is shown to accelerate protein folding by 6-fold and enhance conformational stability by DeltaTm = 2.8 +/- 0.3 degrees C while having no effect on enzymatic activity. The rational use of this and other prosthetic segments could enable chemotherapeutic proteins to survive longer in vivo or retain activity after oral administration.

Kinetics↗

PubMed2003

An electronic effect on protein structure.

The well-known preference of the peptide bond for the trans conformation has been attributed to steric effects. Here, we show that a proline residue with an N-formyl group (H(i-1)-C'(i-1)=O(i-1)), in which H(i-1) presents less steric hindrance than does O(i-1), likewise prefers a trans conformation. Thus, the preference of the peptide bond for the trans conformation cannot be explained by steric effects alone. Rather, an n --> pi* interaction between the oxygen of the peptide bond (O(i-1)), and the subsequent carbonyl carbon in the polypeptide chain (C'(i)) also contributes to this preference. The O(i-1) and C'(i) distance and O(i-1).C'(i)=O(i) angle are especially favorable for such an n --> pi* interaction in a polyproline II helix. We propose that this electronic effect provides substantial stabilization to this and other elements of protein structure.

Amides↗

PubMed2002

Protein prosthesis: a semisynthetic enzyme with a beta-peptide reverse turn.

beta-Amino acids are incorporated into an enzyme by using the method of expressed protein ligation. In the resulting semisynthetic enzyme, an R-nipecotic acid-S-nipecotic acid module replaces Asn113 and Pro114 of ribonuclease A. The semisynthetic enzyme not only retains full catalytic activity but also gains conformational stability. Thus, structural elements can be replaced with foldameric equivalents to endow proteins with more desirable properties.

Catalysis↗

PubMed2002

Semisynthesis of ribonuclease A using intein-mediated protein ligation.

The introduction of non-natural amino acid residues or modules into proteins provides a new means to explore the basis for conformational stability, folding/unfolding behavior, or biological function. We exploited intein-mediated protein ligation to produce a semisynthetic ribonuclease A. Of the 124 residues of RNase A, residues 1-94 were linked to an intein. After expression of the fusion protein and thiol-induced cleavage, the RNase A(1-94) fragment possessed a C-terminal thioester. A peptide identical to the C-terminal residues 95-124 of RNase A (with residue 95 being cysteine) was successfully ligated to that thioester thereby reconstituting full-length wild-type RNase A. In mass spectrometry, this semisynthetic RNase A proved to be undistinguishable from the control protein, namely recombinant wild-type RNase A. Recombinant wild-type RNase A was obtained by expression of RNase A(1-124)-intein fusion protein followed by thiol-induced cleavage and hydrolysis of the thioester. Both proteins showed thermal stabilities (Tm) and catalytic activities comparable to the wild-type enzyme, indicating that both proteins folded properly. These results might serve as basis for the semisynthesis of RNase A variants containing non-natural modules in the aforementioned peptide.

Models, Molecular↗