Cooperativity in natural versus de novo repeat proteins and functional ramifications

Protein folding cooperativity;Repeat proteins;Biophysics

Geiger, Kathryn RachelleBailey, Scott ;

Johns Hopkins University

关键词: Protein folding cooperativity;    Repeat proteins;    Biophysics;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/60230/GEIGER-DISSERTATION-2017.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

Proteins fold and unfold inside living cells. The three-dimension fold of a protein is determined by the order of amino acids in its primary sequence. Folding intermediates are rarified making the cooperativity in protein folding a challenging area of study. Linear repeat proteins make only contacts close in sequence space, thus reducing the number of interacting subunits. One-dimensional Ising models are employed to determine intrinsic and interfacial folding free energies through studying the length-dependence on stability of homopolymeric repeat proteins. Cooperativity in unnatural helical repeat proteins called de novo Helical Repeats (DHRs) is interrogated. These repeat proteins were designed by David Baker’s group using the design principles found in the Rosetta software. Both the sequence and structure of DHRs are dissimilar to any observed natural proteins. DHRs fold cooperatively, but they do so in a novel way. Unlike all previously studied naturally-derived consensus repeat proteins, DHRs have favorable intrinsic energies. As a result, DHRs are extremely fast-folding. These results prove that nature could have partitioned stability in a different way, and offer an opportunity for discussion about the evolution of cooperativity and stability in protein folding.Cooperativity of a naturally-occurring repeat protein, transcription activator-like effectors (TALEs) is also investigated. TALEs bind double stranded DNA one base pair per repeat, and the DNA-binding specificity is determined by two residues in each repeat called repeat-variable diresidues (RVDs). Consensus TALEs (cTALEs) as well as solubilizing capping motifs are designed. Sequence changes of the RVDs affect the stability and cooperativity of cTALE arrays. cTALEs are moderately cooperative, populating several types of partly folded states. Population of partly folded cTALE states are tuned for function. Single molecule total internal reflection fluorescence (smTIRF) microscopy and cell-based assays in S. cerevisiae show that the cTALEs bind DNA and activate transcription similar to naturally-occurring TALEs. Long movies from smTIRF experiments show binding and unbinding kinetics are multi-phasic suggesting conformational heterogeneity in both free and DNA-bound states. cTALE arrays containing enough repeats to form multiple turns around DNA must unfold to populate higher energy ;;open” states which are DNA-binding competent. Binding is initiated through a short lived encounter complex which either disassembles or proceeds to a longer lived DNA-bound ;;locked” state. While this work proves conformational heterogeneity in cTALE binding and unbinding, future work is required to gain insight into the structural details of states involved.