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Title: New tools to study the conformational dynamics of large proteins: A study of FBP-aldolase and other enzymes
Author: Burnley, B. Tom
Current Institution: University of Leeds
Date of Award: 2007
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Proteins are inherently flexible molecules, both in structure and in function. Although multiple conformational changes appear vital for enzyme catalysis, these essential . motions are poorly understood for the majority of systems at present. Escherichia coli Class II fructose 1,6-bisphosphate aldolase (FBP-aldolase) provides an excellent model system for the study of enzyme dynamics as it has been extensively characterised and adopts the (a1~)8 barrel fold. This ubiquitous and versatile architecture represents -10% of all known enzyme structures. Multiple structures of FBP-aldolase have been solved previously, and reveal significant conformational changes. In addition;· sitedirected mutagenesis studies restricting loop flexibility have been shown to reduce catalytic rate, further highlighting the potential importance of conformational dynamics. The work presented here expands these studies by using a novel suite of NMR relaxation experiments to present the first quantified description of the su19-nanosecond dynamics of a protein that exhibits the (a/~)8 barrel.morphology. Experimental studies of this important fold have been restricted due to the size and high helical content that cause highly overlapped 20 spectra. This critically hinders NMR relaxation techniques, preventing the observation of molecular motion at atomic resolution. 20 HSQC based NMR ,sp~troscopy experiments only produce unambiguous rate measurement of less than 40% of residues in FBP-aldolase. Here, a novel sUit~ of Hadamard CO' encoded T1, T2 and NOE pulse sequences is presented· which dramatically reduce spectral crowding to allow the measurement of over 80% of the cross-peaks in FBP-aldolase spectra. The general applicability of these new methods will allow NMR relaxation studies to be conducted on significantly larger protein systems. To apply these Hadamard encoded pulse sequences, and also to assign the backbone amide chemical shifts, 2H, 13C and 15N isotopically enriched FBP-aldolase samples were prepared. Expression and purification protocols were optimised to provide suffiCient material to maximise resonance signal. These samples, in conjunction with multidimensional TROSY experiments, yielded chemical shift assignment for 55% of backbone resonances. This assignment process was aided by the production of seven samples of FBP-aldolase selectiVity labelled for specific amino' acid types. The T10 Tz and NOE relaxation rates were measured for FBP~aldolase at two high-field strengths, 750MHz and 900MHz. The data were quantified using an adapted version ·of the model-free protocol, and revealed important dynamic contributions from the major catalytic loop and the catalytic divalent cation. These experiments were repeated in the presence of the first binding substrate DHAp, which illustrated that the major loop remains flexible to accommodate the binding of the second substrate, G3P. In addition, a new protocol for the comparison of crystal structure pairs based on the distance difference matrix (DDM) is presented. An extensive database of enzyme structure pairs was generated, and the DDM-based methods were used to seek and describe structural deviation. This provided a comprehensive description of the prevalence, magnitude and manner of substrate-induced conformational changes. This study also demonstrates the conformational fluctuations present in enzyme pairs when both structures are solved either in the presence or absence of substrate. The methods developed in this work provide a detailed description of sub-nanosecond dynamics in FBP-aldolase and the conformational changes obseNed for enzymes in general. It is hoped that these novel tools will advance future investigations to further delineate the role of dynamics in enzyme catalysis.
Supervisor: Not available Sponsor: Not available
Qualification Name: UNIVERSITY OF LEEDS, 2007 Qualification Level: Doctoral
EThOS ID:  DOI: Not available