Loop anchor modification causes the population of an alternative native state in an SH3‐like domain

Abstract

Many stably folded proteins are proposed to contain long, unstructured loops. A series of hybrid proteins (EbE1-4) containing the folded scaffold of photosystem I accessory protein E (PsaE), an SH3-like protein, and the 40-residue heme-binding loop of cytochrome b 5 was created to inspect the dependence of thermodynamic and kinetic parameters on the residues at the interface of folded and flexible regions. Compared to the simplest hybrid (EbE1), the chimeras differed by Gly insertions (EbE2, EbE3) or an asymmetric four-residue restructuring of loop termini (EbE4). NMR spectroscopy indicated that the chimeras retained the PsaE topology; native and unfolded state solubilities, however, were affected to varying degrees. Thermal and chemical denaturation experiments revealed that the EbE2 and EbE1 constructs resulted in a modest destabilization of the PsaE core, whereas apparent stability was increased by >5 kJ/mol in EbE4. EbE3 aggregated at mM concentrations and was not studied in detail. EbE4 populated two native states (N1 and N2), which differed by hydrophobic core packing and C-terminal interactions. At room temperature, the population ratio (;3-4:1) favored the state whose spectroscopic properties most resembled those of PsaE (N1). EbE4 also demonstrated altered folding kinetics, displaying multiple slow phases related to the population of intermediates and possibly N2. It was concluded that loop anchors can affect protein properties, including stability, via short-range effects on local structure and long-range communication with the packed hydrophobic core. Modification of the attachment points appears to be a possible stepping stone in the transition from one three-dimensional structure to another.

Structural propensities in the heme binding region of apocytochrome b5. I. Free peptides

“…18,19 The influence of the heme binding loop on its supporting scaffold has been investigated by substitution for a flexible loop in photosystem I accessory protein E (PsaE). 19,20 Chemical and thermal denaturation of the chimeric proteins suggests that the thermodynamic cost of closing the H2-H5 loop in apocyt b 5 need not be high. The presence of residual structure in H2 and elsewhere may explain values lower than the Gaussian estimate.…”

Structural propensities in the heme binding region of apocytochrome b5. II. Heme conjugates

“…In prior work, chimeric constructs consisting of the cyt b 5 heme binding loop (*40 residues encompassing H2-H3-b5-H4-H5) grafted onto an alternate scaffold protein, an SH3-like domain, were prepared to establish the free energy penalty of carrying this particular intrinsically disordered region. 12,13 With a family of four closely related chimeras and their denaturation behavior, it was found that the energetic cost depended strongly on the way in which the loop-scaffold interface was designed. The four chimeras did not bind heme specifically and emphasized the importance of correctly configuring the chain trajectory from and back into the folded scaffold.…”