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PubMed1987

Is thermally denatured protein unfolded? The example of alpha-lactalbumin.

The degree of unfolding of various forms of human and bovine alpha-lactalbumin was characterized by the chromatographically determined distribution coefficient. Molecular size increases as follows: native protein approximately equal to apo form less than acid form much less than thermally unfolded form approximately equal to guanidine hydrochloride-unfolded protein.

Animals↗
PubMed1995

Divergent effects of ATP on the binding of the DnaK and DnaJ chaperones to each other, or to their various native and denatured protein substrates.

Using the native proteins lambda P, lambda O, delta 32, and RepA, as well as permanently unfolded alpha-carboxymethylated lactalbumin, we show that DnaK and DnaJ molecular chaperones possess differential affinity toward these protein substrates. In this paper we present evidence that the DnaK protein binds not only to short hydrophobic peptides, which are in an extended conformation, but also efficiently recognizes large native proteins (RepA, lambda P). The best substrate for either the DnaK or DnaJ chaperone is the native P1 coded replication RepA protein. The native delta 32 transcription factor binds more efficiently to DnaJ than to DnaK, whereas unfolded alpha-carboxymethylated lactalbumin or native lambda P binds more efficiently to DnaK than to the DnaJ molecular chaperone. The presence of nucleotides does not change the DnaJ affinity to any of the tested protein substrates. In the case of DnaK, the presence of ATP inhibits, while a nonhydrolyzable ATP analogues markedly stimulates the binding of DnaK to all of these various protein substrates. ADP has no effect on these reactions. In contrast to substrate protein binding, DnaK binds to the DnaJ chaperone protein in a radically different manner, namely ATP stimulates whereas a nonhydrolyzable ATP analogue inhibits the DnaK-DnaJ complex formation. Moreover, the DnaKc94 mutant protein lacking 94 amino acids from its C-terminal domain, which still possesses at ATPase activity and forms a transient complex with protein substrates, does not interact with DnaJ protein. We conclude that the DnaK-ADP form, derived from ATP hydrolysis, possesses low affinity to the protein substrates but can efficiently interact with DnaJ molecular chaperone.

Adenosine Diphosphate↗
PubMed1991

ATP-induced conformational transition of denatured proteins.

Although the conformational change occurring in proteins upon ATP binding is important in many biological reactions, the mechanism by which ATP binding induces the conformational change is unknown. We found that ATP induces acid-unfolded (pH 2) ferricytochrome c or apomyoglobin to adopt a compact structure with a significant amount of alpha-helix and increased hydrophobicity. A very similar conformational transition was observed at neutral pH for an amphiphilic model polypeptide. The effectiveness of various adenine nucleotides in inducing the conformational transition was found to be proportional to their phosphate group contents, i.e., adenosine tetraphosphate greater than ATP greater than ADP greater than AMP. These results should be important when considering the mechanism of the ATP-induced conformational change in proteins during various biological reactions.

Adenine Nucleotides↗
PubMed1975

Reversible inactivation of pancreatic deoxyribonuclease A by sodium dodecyl sulfate. Removal of COOH-terminal residues from the denatured protein by carboxypeptidase A.

In the course of experiments on the role of the COOH-terminal residues in pancreatic deoxyribonuclease, we undertook to ascertain whether the presence of sodium dodecyl sulfate would render the normally unavailable terminus susceptible to hydrolysis by carboxypeptidase A. When DNase A is dissolved in 0.005% sodium dodecyl sulfate the protein becomes enzymically inactive when assayed against DNA in the same sodium dodecyl sulfate concentration. The loss of activity caused by treatment with sodium dodecyl sulfate for 1 hour at 45 degrees can be fully restored if the detergent-containing solution is diluted 10-fold into 6 M guanidinium chloride and then 10-fold into a pH 7.0 buffer, 10 mM in CaCl2, prior to a 100-fold dilution for assay. The presence of Ca2+ is essential for the refolding process. If the same degree of dilution is made into sodium dodecyl sulfate-free buffer without the guanidinium chloride step, there is very little reversal of the inactivation. An almost complete loss of regenerable activity is caused by 1 hour of digestion by carboxypeptidase at 45 degrees in the presence of 0.03% sodium dodecyl sulfate. Although up to 6 amino acid residues can be removed from the COOH terminus, the loss of activity can be correlated with the removal of either 1 or 2 amino acid residues (-Leu-Thr) from the COOH-terminal sequence. Thus, DNase A is one of the several enzymes in which residues at the COOH terminus are essential to the active conformation. If the enzyme minus 2 to 6 terminal residues was mixed with a 15-residue COOH-terminal peptide (obtained by cyanogen bromide cleavage), only about 2% activity could be regenerated.

Amino Acid Sequence↗