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A general method for the quantitative analysis of functional chimeras: Applications from site-directed mutagenesis and macromolecular association

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720–3206, USA

Reprint requests to: Dr. Jack F. Kirsch, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720–3206, USA; e-mail: jfkirsch{at}uclink4.berkeley.edu; fax: (510) 642-6368.

Two new parameters, I and C, are introduced for the quantitative evaluation of functional chimeras: I (impact) and C (context dependence) are the free energy difference and sum, respectively, of the effects on a given property measured in forward and retro chimeras. The forward chimera is made by substitution of a part "a" from ensemble A into the analogous position of homologous ensemble B (S_{B A}). The C value is a measure of the interaction of the interrogated position with its surroundings, whereas I is an expression of the quantitative importance of the probed position. Both I and C vary with the evaluated property, for example, kinetics, binding, thermostability, and so forth. The retro chimera is the reverse substitution of the analogous part "b" from B into A, S_{A B}. The I and C values derived from original data for forward and retro mutations in aspartate and tyrosine aminotransferase, from literature data for quasi domain exchange in oncomodulin and for the interaction of Tat with bovine and human TAR are evaluated. The most salient derived conclusions are, first, that Thr 109 (AATase) or Ser 109 (TATase) is an important discriminator for dicarboxylic acid selectivity by these two enzymes (I < -2.9 kcal/mol). The T109S mutation in AATase produces a nearly equal and opposite effect to S109T in TATase (C < 0.4 kcal/mol). Second, an I value of 5.5 kcal/mol describes the effects of mirror mutations D94S (site 1) and S55D (site 2) in the Ca²⁺ binding sites of oncomodulin on Ca²⁺ affinity. The second mirror set, G98D (site 1) and D59G (site 2), yields a smaller impact (I = -3.4 kcal/mol) on Ca²⁺ binding; however, the effect is significantly more nearly context independent (C = -0.6 versus C = -2.7 kcal/mol). Third, the stem and loop regions of HIV and BIV TAR are predominantly responsible for the species specific interaction with BIV Tat_65–81 (I = -1.5 to -1.6 kcal/mol), whereas I = 0.1 kcal/mol for bulge TAR chimeras. The C values are from -0.3 to -1.2 kcal/mol. The analysis described should have important applications to protein design.

Keywords: Aminotransferase; chimera; context dependence; macromolecular interactions; protein/genetic engineering; oncomodulin; Tat/TAR

Abbreviations: PLP, pyridoxal-5'-phosphate • -KG, -ketoglutarate • AATase, aspartate aminotransferase (EC 2.6.1.1) • TATase, tyrosine aminotransferase (EC 2.6.1.5) • MDH, malate dehydrogenase • OAA, oxaloacetate • PMP, pyridoxamine-5'-phosphate • HO-HxoDH, 2-hydroxyisocaproate (i.e. 2-hydroxy-4-methyl-pentanoate) dehydrogenase (EC 1.1.1.-) • Hca, hydrocinnamate • HIV, human immunodeficiency virus • BIV, bovine immunodeficiency virus • TAR, trans-activation response element • Tat, trans-activator of transcription • HEX, a hexamutant of AATase with the following six substitutions V39L/K41Y/T47I/N69L/T109S/N297S

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