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

Two new parameters, I and C, are introduced for the quantitative evaluation of functional chimeras: I (impact) and C (context dependence) are the foe energy difference and sum, respectively, of the effects on a given property measured in forward and retro chimeras. The forward chimera is made by sub stitution of a part "a" from ensemble A into the analogous position of homo logous ensemble B (SB -->A). The C value is a measure of the interaction of the interrogated position with its surroundings, whereas I is an expressio n of the quantitative importance of the probed position. Both I and C vary with the evaluated property, for example, kinetics, binding, thermostabilit y, and so forth. The retro chimera is the reverse substitution of the analo gous part "b" from B into A, SA -->B. The I and C values derived from origi nal data for forward and retro mutations in aspartate and tyrosine aminotra nsferase, from literature data for quasi domain exchange in oncomodulin and for the interaction of Tar with bovine and human TAR are evaluated. The mo st salient derived conclusions are, first, that Thr 109 (AATase) or Ser 109 (TATase) is an important discriminator for dicarboxylic acid selectivity b y these two enzymes (I < -2.9 kcal/mol). The T109S mutation in AATase produ ces 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 mutat ions D94S (site 1) and S55D (site 2) in the Ca2+ binding sites of oncomodul in on Ca2+ affinity. The second mirror set, G98D (site 1) and D59G (site 2) , yields a smaller impact(l = -3.4 kcal/mol) on Ca2+ binding; however, the effect is significantly more nearly context independent (C = -0.6 versus C = -2.7 kcaymol). Third, the stem and loop regions of HIV and BIV TAR are pr edominantly responsible for the species specific interaction with BIV Tat(6 5-81) (I = -1.5 to -1.6 kcal/mol), whereas I = 0.1 kcal/mol for bulge TAR c himeras. The C values are from -0.3 to -1.2 kcal/mol. The analysis describe d should have important applications to protein design.