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

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 microM 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 ( approximately 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.

A buried polar residue in the hydrophobic interface of the coiled-coil peptide, GCN4-p1, plays a thermodynamic, not a kinetic role in folding.

The hydrophobic interfaces of coiled-coil proteins and peptides are typically interspersed with buried polar residues. These polar residues are known to be important for defining oligomeric specificity and chain orientation in coiled-coil formation; however, their effects on the folding/assembly reaction have not been investigated. The commonly studied 33-residue dimeric leucine zipper peptide, GCN4-p1, contains a single polar Asn in the center of the hydrophobic interface at position 16. Peptides containing either a valine or an alanine replacement at this position, N16V and N16A, respectively, were studied in order to investigate both the thermodynamic and kinetic roles of the buried polar side-chain on the folding of GCN4-p1. Equilibrium sedimentation confirmed that both the N16V and N16A mutations reduce the dimeric specificity of GCN4-p1, leading to the population of both dimers and trimers in the absence of denaturant. Guanidine hydrochloride-induced equilibrium unfolding of the mutant peptides demonstrated that N16V is more stable than the wild-type sequence, while the N16A peptide is moderately destabilized. Comparison of the refolding reactions indicate that Asn16 is not involved in the rate-limiting association step leading to the native dimer; only the unfolding reaction is sensitive to the mutations. More complex unfolding kinetics for both peptides at high peptide concentrations can be attributed to the presence of trimers in the absence of denaturant. These results show that the role of buried polar residues in leucine zipper peptides can be primarily thermodynamic; subunit exchange reactions can be controlled by the stability of the native coiled coil and its influence on the unfolding/dissociation process.

Insertion of the cytochrome b5 heme-binding loop into an SH3 domain. Effects on structure and stability, and clues about the cytochrome's architecture.

Under native conditions, apocytochrome b(5) exhibits a stable core and a disordered heme-binding region that refolds upon association with the cofactor. The termini of this flexible region are in close proximity, suggesting that loop closure may contribute to the thermodynamic properties of the apocytochrome. A chimeric protein containing 43 residues encompassing the cytochrome loop was constructed using the cyanobacterial photosystem I accessory protein E (PsaE) from Synechococcus sp. PCC 7002 as a structured scaffold. PsaE has the topology of an SH3 domain, and the insertion was engineered to replace its 14-residue CD loop. NMR and optical spectroscopies showed that the hybrid protein (named EbE1) was folded under native conditions and that it retained the characteristics of an SH3 domain. NMR spectroscopy revealed that structural and dynamic differences were confined near the site of loop insertion. Variable-temperature 1D NMR spectra of EbE1 confirmed the presence of a kinetic unfolding barrier. Thermal and chemical denaturations of PsaE and EbE1 demonstrated cooperative, two-state transitions; the stability of the PsaE scaffold was found only moderately compromised by the insertion, with a DeltaT(m) of 8.3 degrees C, a DeltaC(m) of 1.5 M urea, and a DeltaDeltaG degrees of 4.2 kJ/mole. The data implied that the penalty for constraining the ends of the inserted region was lower than the approximately 6.4 kJ/mole calculated for a self-avoiding chain. Extrapolation of these results to cytochrome b(5) suggested that the intrinsic stability of the folded portion of the apoprotein reflected only a small detrimental contribution from the large heme-binding domain.

Proximal influences in two-on-two globins: effect of the Ala69Ser replacement on Synechocystis sp. PCC 6803 hemoglobin.

The cyanobacterium Synechocystis sp. PCC 6803 (S6803) expresses a two-on-two globin in which His46 (distal side) and His70 (proximal) function as heme iron axial ligands. His46 can be displaced by O2, CO, and CN-, among others, whereas His70 is not labile under native conditions. The residue preceding the proximal histidine has been implicated in controlling globin axial ligand reactivity; the details of the mechanism, however, are not well understood, and little information exists for bis-histidyl hexacoordinate proteins. In many vertebrate hemoglobins and in the Synechocystis protein, the position is occupied by an alanine, whereas, in myoglobins, it is a serine involved in an intricate hydrogen-bond network. We examined the role of Ala69 in S6803 hemoglobin through the effects of an Ala --> Ser replacement. The substitution resulted in minor structural perturbations, but the response of the holoprotein to temperature-, urea-, and acid-induced denaturation was measurably affected. Enhanced three-state behavior was manifested in the decoupling of heme binding and secondary-structure formation. Urea-gradient gel experiments revealed that the stability of the apoprotein was unchanged by the replacement and that a slight alteration of the folding kinetics occurred in the holoproteins. Cyanide-binding experiments were performed to assess trans effects. The apparent rate constant for association decreased 2-fold upon Ala69Ser replacement. This deceleration was attributed to a change in the lifetime of a state containing a decoordinated His46. The results demonstrated that, as in vertebrate globins and leghemoglobin, proximal influences operate to determine fundamental dynamic and thermodynamic properties of the protein.