Abstract
Non-local hydrogen bonding interactions between main chain amide hydrogen atoms and polar side chain acceptors that bracket consecutive βα or αβ elements of secondary structure in αTS from E. coli, a TIM barrel protein, have previously been found to contribute 4–6 kcal mol−1 to the stability of the native conformation. Experimental analysis of similar βα-hairpin clamps in a homologous pair of TIM barrel proteins of low sequence identity, IGPS from S. solfataricus and E. coli, reveals that this dramatic enhancement of stability is not unique to αTS. A survey of 71 TIM barrel proteins demonstrates a 4-fold symmetry for the placement of βα-hairpin clamps, bracing the fundamental βαβ building block and defining its register in the (βα)8 motif. The preferred sequences and locations of βα-hairpin clamps will enhance structure prediction algorithms and provide a strategy for engineering stability in TIM barrel proteins.
Highlights
The8, TIM barrel is one of the most common folds in biology, supporting a myriad of catalytic functions essential to life [1]
Experimental analysis of ba-hairpin clamp interactions in two TIM barrel proteins The generality of the potent hairpin clamps found in alpha subunit of tryptophan synthase (aTS) was tested by mutational analysis of ba-hairpin clamps in two homologous TIM barrel proteins with low sequence identity (,30%) to aTS and to each other. sIGPS (Figure 1A) and eIGPS (Figure 1B), each contain three ba-hairpin clamps (Figure 1C and 1D), some of which are conserved in location with those in aTS and others between sIGPS and eIGPS
The b1a1 clamp is observed in aTS and eIGPS, the b2a2 clamp only appears in sIGPS, the b3a3 clamp is observed in all three proteins, and the b7a7 clamp is observed in sIGPS
Summary
The (ba), TIM barrel is one of the most common folds in biology, supporting a myriad of catalytic functions essential to life [1]. Experimental [2] and bioinformatics [1,3,4] analyses of TIM barrel proteins have led to the conclusion that a pair of adjacent parallel b-strands and the intervening anti-parallel a-helix, i.e., the bab module, serve as the minimal unit of stability. Gene duplication of this elemental bab building block into higher-order structures has been suggested to result in several common barepeat structures, including the TIM barrel, Rossman, flavodoxin and leucine-rich folds [3]. Mutational analysis has shown that these non-covalent interactions usually contribute modestly, typically in the range of 1– 2 kcal mol, to the stability of their resident proteins [11,12,13,14]
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