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Research has been conducted on the folding kinetics of the real proteins. The putative relationship between the nonspecific compaction of the unfolded state and protein folding kinetics has been investigated via the small-angle X-ray scattering and circular dichroism spectroscopy and the results are discussed.

The kinetics and thermodynamics of common-type acylphosphatase and muscle acylphosphatase have been studied using spectroscopic probes to monitor the folding and unfolding process. Results show that both proteins fold via a two-fold mechanism. Under all conditions examined, common-type acylphosphatase retains a lower conformational stability as compared to the homologous muscle form. Folds also occur more quickly in common-type acylphosphatase, indicating that conformational stability and the folding rate are not correlated as suggested by previous research.

A study was conducted on the conformational stability of muscle acylphosphatase as a function of temperature, pH and urea concentration. The findings indicate that acylphosphatase is a relatively unstable globular protein with low midpoints for both thermal and chemical denaturation. The protein's maximum stability is pegged at new ph 5.5. Moreover, its conformational stability increases at low pH values, implying that the overall dimensions of the unfolded state are influenced by the number of charges within the polypeptide chain.

Different spectroscopic techniques were utilized to study the kinetic, structural, and stability features of a number of muscle acylphosphatase mutants in the 15-21 and 42-45 loop regions. Results have shown that 42-45 loop has an important role in the stabilization of the acylphosphatase active binding site and that its integrity is a basic requisite for optimum catalytic performance.

The relationship between Asn41, Thr42 and Thr46 residues in muscle acylphosphatase and Arg23, a residue found in the main substrate binding site, was investigated in five gene mutants of the muscular form using oligonucleotide-directed mutagenesis. The results revealed well-constrained native-like secondary and tertiary structures. All mutants exhibited stability except the Thr42 mutant which decreased stability under increased temperatures and acidity. Data also suggested a role for Asn41 in enzyme catalysis.

The binding properties of single-stranded and double-stranded DNA, ATP, heparin, and heparan sulfate, which are representative of three major nonproteinaceus biological polyanions, are examined. The differences in affinity between native and aggregated acylphosphatase with heparin suggest that amyloid fibrils can behave as polyelectrolytes, interacting strongly with other polyelectrolytes bearing the opposite charge.

A study is conducted to examine the folding process of the acylphosphatase from Sulfolobus solfataricus and compared to other proteins from the same family. The results indicate that an ensemble of partially folded or misfold species form rapidly on the submillisecond time scale after initiation of folding.

The relative importance of net charge, as opposed to the other factors, on protein aggregation and amyloidogenicity is evaluated with the help of two homologus proteins. The difference in net charge between the two-wild type proteins contributes to 20-25 percent of the difference in their aggregation rates.