Using chimeric immunity proteins to explore the energy landscape for alpha-helical protein folding.
To address the role of sequence in the folding of homologous proteins, the folding and unfolding kinetics of the all-helical bacterial immunity proteins Im2 and Im9 were characterised, together with six chimeric derivatives of these proteins. We show that both Im2 and Im9 fold rapidly (k(UN)(H(2)O)) approximately 2000 s(-1) at pH 7.0, 25 degrees C) in apparent two-state transitions, through rate-limiting transition states that are highly compact (beta(TS)0.93 and 0.96, respectively). Whilst the folding and unfolding properties of three of the chimeras (Im2 (1-44)(Im9), Im2 (1-64)(Im9 )and Im2 (25-44)(Im9)) are similar to their parental counterparts, in other chimeric proteins the introduced sequence variation results in altered kinetic behaviour. At low urea concentrations, Im2 (1-29)(Im9) and Im2 (56-64)(Im9) fold in two-state transitions via transition states that are significantly less compact (beta(TS) approximately 0.7) than those characterised for the other immunity proteins presented here. At higher urea concentrations, however, the rate-limiting transition state for these two chimeras switches or moves to a more compact species (beta(TS) approximately 0.9). Surprisingly, Im2 (30-64)(Im9) populates a highly collapsed species (beta(I)=0.87) in the dead-time (2.5 ms) of stopped flow measurements. These data indicate that whilst topology may place significant constraints on the folding process, specific inter-residue interactions, revealed here through multiple sequence changes, can modulate the ruggedness of the folding energy landscape.
School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
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Ferguson
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Li
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Capaldi
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Kleanthous
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Radford
D000595:Amino Acid Sequence; D001419:Bacteria / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D003087:Colicins / Q000737:chemistry*; D004734:Energy Metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D003087:Colicins; D011993:Recombinant Fusion Proteins; C007104:colicin immunity proteins