Biomedical subjects

H Reiersen

Publications and source records attributed to H Reiersen.

7 recordsLinked to original sources

PubMed2001

The hunchback and its neighbours: proline as an environmental modulator.

The unique ability of Pro or Pro-rich repeats to affect the stability and function of proteins has recently been highlighted by biophysical studies on fragments from prions, signalling domains and muscle proteins. Pro-rich regions have been observed to either occupy disordered states or adopt various helical structures; some are also able to undergo an environmental-dependent transformation between these states. Such a transformation could explain some of the inherent functional properties of the parent proteins and, additionally, can be efficiently exploited to generate novel temperature- and pH-switches in more conventional globular proteins.

Amino Acid Sequence↗

PubMed2000

Sodium sulphate reactivates a protein A minidomain with a short elastin beta-turn.

Elastin polymer sequences derived from muscle exhibit temperature and salt-induced reversible contractions and expansions. A folded and contracted beta-turn helical elastin structure is stabilised by increased intramolecular hydrophobic interactions. We have generated a switchable binding protein by inserting a typical elastin turn sequence, GVPGVG, between the two IgG-binding helices of an engineered globular minidomain from Protein A. This mutant showed increased binding of IgG-molecules compared to the wild-type sequence in the presence of sodium sulphate, as measured by surface plasmon resonance spectroscopy. Analysis by circular dichroism revealed a salt-induced folding of the mutant minidomain to a native type I beta-turn, likely stabilised as a result of the two interacting dehydrated valines across the beta-turn. Since sodium sulphate can be successfully used to regulate the folding/unfolding or binding/dissociation of this minidomain, this suggests an alternative protein purification method.

Amino Acid Sequence↗

PubMed2000

Trifluoroethanol may form a solvent matrix for assisted hydrophobic interactions between peptide side chains.

Several models for interactions between trifluoroethanol (TFE) and peptides and proteins have recently been proposed, but none have been able to rationalize the puzzling observations that on the one hand TFE can stabilize some hydrophobic interactions in secondary structures, but on the other can also melt the hydrophobic cores of globular proteins. The former is illustrated in this paper by the effect of TFE on a short elastin peptide, GVG(VPGVG)(3), which forms type II beta-turns stabilized by hydrophobic interactions between two intra-turn valine side chains. This folding, driven by increasing the entropy of bulk water, is stimulated in TFE-water mixtures and/or by raising the temperature. To explain these apparently contradictory observations, we propose a model in which TFE clusters locally assist the folding of secondary structures by first breaking down interfacial water molecules on the peptide and then providing a solvent matrix for further side chain--side chain interactions. This model also provides an explanation for TFE-induced transitions between secondary structures, in which the TFE clusters may redirect non-local to local interactions.

Elastin↗

PubMed1999

An engineered minidomain containing an elastin turn exhibits a reversible temperature-induced IgG binding.

A two-helix version of the triple alpha-helical staphylococcal Protein A, previously shown to retain the Fc binding properties of protein A, has been engineered to contain an elastin sequence, GVPGVG, within the inter-helix turn. The original type I beta-turn was replaced with a beta-turn from the muscle protein elastin, which has an inverse temperature-induced folding transition. These "elastin mutants" had lost their helical structure, as measured by circular dichroism (CD), and exhibited a lower stability than the wild-type domains (T(m) reduced by about 48 degrees C) in 30% trifluoroethanol. For the wild-type domains, the amount of alpha-helix and the binding affinity for Fc decreased as the temperature was increased. In contrast, although the starting affinity was lower for the disulfide elastin-turn mutant, it exhibited a 21-fold improvement in affinity over the same temperature range. The melting curve for the elastin-turn minidomain showed cooperative behavior, as measured by the increase in CD-amplitude at 222 nm. The observed CD behavior is consistent with the formation of a type I beta-turn, exhibiting similar DeltaH and DeltaS values to those seen previously for short elastin peptides [Reiersen, H., Clarke, A. R., and Rees, A. R. (1998) J. Mol. Biol. 283, 255-264], and accounting for the increase in on-rate. This demonstrates that, when inserted into a stable globular protein, short elastin sequences have the ability to modify local structure and activity, by operating as temperature modulated switches.

Amino Acid Sequence↗

PubMed1998

Short elastin-like peptides exhibit the same temperature-induced structural transitions as elastin polymers: implications for protein engineering.

Elastin is a major protein component of the vascular wall and is responsible for its unusual elastic properties. Polymers of its repeating VPGVG sequences have been synthesised and shown to exhibit an inverse temperature transition where, as temperature rises, the polymer collapses from an extended chain to a beta-spiral structure with three VPGVG units per turn, each pentamer adopting a type II beta-turn conformation. These studies, however, have not established whether the temperature-driven conformational change is an intrinsic property of the individual pentameric sequences or a global, co-operative effect of many pentamers within the beta-spiral structure. Here, we examine by circular dichroism the behaviour of elastin-like peptides (VPGVG)n, where n varies between 1 and 5. Remarkably, we find that all lengths of peptide undergo an extended left and right arrow beta-turn transition with increasing temperature, suggesting that the induction of the beta-spiral occurs at the level of single pentameric units. The origin of this effect is a positive DeltaS term for the transition. At 35 degreesC, the average transition midpoint temperature, the value of TDeltaS is about 15 kcal mol-1. With larger oligomers (n=3), there is only a modest rise in DeltaS, suggesting that the dominant entropic effect resides within the monomer and that interactions between these units make only a small contribution to the energetics of the transition. Charges at the termini, and residue replacements or additions, regulate the transitions for the short peptides in a manner similar to that observed for the longer polymers. The behaviour of the same peptides in trifluoroethanol and SDS solutions is consistent with formation of the beta-turn being driven by interactions between non-polar groups. The significance of this behaviour for the rational design of temperature-induced responses in proteins is discussed.

Amino Acid Sequence↗

PubMed1994

The inactivation of sheep liver sorbitol dehydrogenase by pyrophosphate and some analogous metal chelators.

Pyrophosphate and several other metal chelators are shown to inactivate sheep liver sorbitol dehydrogenase. Pyrophosphate, tripolyphosphate, and some bisphosphonates inactivate the enzyme by saturation kinetics involving the formation of a reversible complex. A mechanism for the pyrophosphate-mediated inactivation of sorbitol dehydrogenase is proposed. Steady-state kinetics show that pyrophosphate does not compete with sorbitol for binding to the catalytic zinc atom or with NAD for binding to the anion binding site. The latter is supported by the formation of an E-NAD-pyrophosphate (PPi) complex and by the noncompetitive protection of NADH against inactivation. The rate of enzyme inactivation by pyrophosphate increases with decreasing pH. The pH dependence of the inactivation indicates that a group with a pKa of 6.9 in the free enzyme and in the enzyme-PPi complex is involved. As several zinc-binding reversible inhibitors do not afford protection against pyrophosphate inactivation, the pKa values obtained are considered not to refer to the ionization of the zinc-bound water molecule, but are tentatively suggested to be those of an active site histidine residue. Protection and reactivation by Zn2+ ions indicate that enzyme inactivation results from the loss of the catalytic zinc atom.

Animals↗

PubMed1993

Affinity labelling of sorbitol dehydrogenase from sheep liver with alpha-bromo-beta-(5-imidazolyl)propionic acid.

The metal-directed alkylating agent DL-alpha-bromo-beta-(5- imidazolyl)propionic acid (BrImPpOH) is shown to be an affinity-labelling reagent for sheep liver sorbitol dehydrogenase (SDH). As previously found for horse liver alcohol dehydrogenase (ADH), it modifies a cysteine ligand to the active-site zinc. In this case it is selectively incorporated (over 90%) at Cys43 in each of the four polypeptide chains/protomers of sheep liver SDH. Incorporated reagent and residual activity correlated. The first order inactivation constant, K2, and KEI, the dissociation constant for SDH and BrImPpOH, have been determined at different pH. The reactivity of BrImPpOH for SDH is higher than that for horse liver and yeast ADH. The protection of SDH against BrImPpOH inactivation by buffers and other molecules shows some similarities to that with horse liver ADH. However, sheep liver SDH bound BrImPpOH, imidazole and phosphate ions much weaker than liver ADH. The pKa values from the plot of log (k2/KEI) against pH are approximately 7.0 and 8.8-8.9. The former pKa value probably represents ionization of an imidazole group and the latter the zinc/water ionization in SDH. These pKa values are similar to those found for horse liver ADH. They are apparently not noticeably influenced by a second cysteine ligand in liver ADH being replaced by a proposed glutamic acid residue as a ligand to the catalytic zinc in SDH. The plot of logk2 against pH shows pKa values around 7.0 and 9.2 for the SDH-BrImPpOH-complex. The pKa of 7.0 is the same as for log(k2/KEI), and indicates no significant perturbation due to the binding of BrImPpOH to SDH. The pKa around 9.2 indicates perturbation of the zinc/water ionization or the ionization of Cys43.

Affinity Labels↗