Design, creation, and characterization of a stable,monomeric triosephosphate isomerase.

Abstract

Protein engineering on trypanosomal triosephosphate isomerase (TIM) converted this oligomeric enzyme into a stable, monomeric protein that is enzymatically active.Wild-type TIM consists of two identical subunits that form a very tight dimer involving interactions of 32 residues of each subunit. By replacing 15 residues of the major interface loop by another 8-residue fragment, a variant was constructed that is a stable and monomeric protein with TIM activity. The length, sequence, and conformation of the designed fragment were suggested by extensive modeling.The (,B/a)g protein fold, commonly known as the triosephosphate isomerase (TIM) fold, seems to be nature's favorite choice of a scaffold for enzymatic activities as %20 different enzymes have, so far, been shown to have this fold (1, 2). The 3-strands form an internal 1-barrel consisting of 8 parallel ,3-strands (strands 1-8), covered on the outside by 8 a-helices (helices [1][2][3][4][5][6][7][8]. The connections between 3-strands and the subsequent a-helices are referred to as loops 1-8. The side chains of some of these loop residues form the active site of the TIM barrel enzymes and novel active sites can therefore theoretically be constructed at the C terminus of the ,/strands without altering the overall fold. Our goal is to design activities on this (,3/a)8 framework. Interestingly, most of the known (/3/a)8 proteins are part of homo-or heterooligomers. For our studies, TIM from Trypanosoma brucei was chosen as the model protein because the crystal structure is known at 1.83 A resolution (3). TIM forms very stable dimers with high catalytic activity. These properties have hindered the accurate measurements of the activity of individual monomers; the monomers have been postulated to be inactive in both renaturation (4, 5) and mutagenesis (6) studies, whereas matrix-bound monomers have been reported to retain activity (7). No allosteric control or cooperativity has been found between the two subunits (8, 9). The Kd of the TIM dimer is not known, but based on the low protein concentration in the activity assay, and assuming that the monomer is much less active than the dimer, then the Kd value can be estimated to be <10 pM. The first step in our protein engineering scheme has been to convert the dimeric TIM to a monomeric protein to (i) facilitate the subsequent design of active sites, (ii) examine the functional and structural importance of dimer formation, and (iii) have the principal possibility to obtain an active monomer. Only few such mutagenesis experiments of other proteins have been described. One example is the protein hormone insulin, in which it has been shown that single and double amino acid alterations of interface residues led to considerable variations in the association of the insulin monomers (10). The dimeric ACro DNA binding protein has been converted to a stable monomer that binds to DNA (11). Both cases involve small nonenzyme proteins on the order of 60 residues and insulin is, in fact, functional as a monomer. Single amino...

Conformational stability of dimeric proteins: Quantitative studies by equilibrium denaturation

“…A stable, monomeric form of the X Cro DNA binding protein that was unable to associate into a dimer has been engineered by rather extensive rearrangement of the subunit interface to prevent productive interactions (Mossing & Sauer, 1990). A similar scheme produced an active, monomeric form of triosephosphate isomerase (Borchert et al, 1994). Surprisingly, both monomeric Cro and the isomerase had apparently higher stabilities, based on an increased T, of denaturation by 13" and 3", respectively (compared to the corresponding wild-type dimer).…”

An interface point‐mutation variant of triosephosphate isomerase is compactly folded and monomeric at low protein concentrations

“…3A) as described previously for mono-TIM [8]. Since the protein concentration of the sample is approximately 1 mg/ml, the majority of the variant protein is dissociated into monomer.…”

“…The Kd of the wild-type protein has been estimated to be below 10 -~1 M [8]. It has been shown that the folding pathway of the TIM-dimer can be described by a consecutive first-order folding and second-order association reaction scheme assuming inactive monomers [7].…”

“…Because of these interdigitating loops at the dimer interface, it is not a priori obvious that the individual wild-type subunits will have a compactly folded structure by themselves. Previously, we have shown that the 15 residues of loop-3 of trypanosomal TIM (wtTIM) can be replaced by an eight residue loop, in such a way that a stable, monomeric protein (monoTIM) is obtained [8]. The crystal structure of monoTIM has been reported [9].…”

“…Loop-3 is a long loop which protrudes 13 A out of the bulk of the monomer and docks into a narrow pocket close to the active site of the other subunit. This loop is very important for the stability of the dimer since 80% of the intersubunit atom atom contacts involve atoms of loop-3 [8]. The narrow pocket is shaped by loop-1 and loop-4.…”