Stability of dimeric retroviral proteases.
The determination of dimer stabilities for the retroviral proteases has proved more challenging than anticipated, but it is a tractable problem when careful attention is made to potential interferences. For investigations of retroviral proteases not yet characterized, the fundamentally rigorous sedimentation equilibrium and other biophysical techniques may yet provide useful Kd values. They are preferable to the indirect methods emphasized in this chapter but nevertheless should be coupled with basic considerations such as recovery of activity at the end of an experiment and the relevance of values obtained to other situations. In the likely event that nanomolar Kd values are encountered in new investigations, the assay techniques provide the most readily available methods for many laboratories. Because these methods are sensitive to anything that affects enzyme activity, the use of complementary methods to verify dimerization constants is imperative. Inactivating reactions not due to monomer formation should be explored, and the potential impact of those reactions on the constants being measured should be estimated. Most of the Kd and dimerization rate data available for retroviral proteases are obtained with the HIV-1 protease, with each investigator choosing methods and solvent conditions different from the others. The confusing diversity of results should be the impetus for a direct comparison of methods for the identification of the sources of differences. If more comprehensive and rigorous measures of the kinetics and thermodynamics of subunit aggregation are obtained, they might be coupled with the large volume of detailed structural data accumulating for this class of protein to provide insights into more general problems of protein-folding chemistry.
Biological Chemistry Department, Merck Research Laboratories, West Point, Pennsylvania 19486.
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Darke
D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D001665:Binding Sites; D005470:Fluorometry; D016333:HIV Protease / Q000737:chemistry; D017320:HIV Protease Inhibitors / Q000494:pharmacology; D015497:HIV-1 / Q000201:enzymology; D015498:HIV-2 / Q000201:enzymology; D007700:Kinetics; D008970:Molecular Weight; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D055503:Protein Multimerization; D012190:Retroviridae / Q000201:enzymology*; D012191:Retroviridae Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry*
D017320:HIV Protease Inhibitors; D012191:Retroviridae Proteins; D016282:Aspartic Acid Endopeptidases; D016333:HIV Protease; C065184:p16 protease, Human immunodeficiency virus 2