| In a large number of proteins, it appears that segments of polypeptide chains resist folding into stable secondary structure. These intrinsically disordered regions (IDRs), which have a higher percentage of polar residues compared to ordered regions, are also found in binding pockets of heme proteins. Disorder and structural flexibility are eliminated once the heme is incorporated in the apoprotein. Partial pre-organization of the heme binding site is expected to be advantageous: it facilitates heme insertion and provides a mechanism for adjusting binding specificity and affinity. To test the binding and folding of these IDR regions, experiments were performed on heme-binding proteins and on peptides from IDRs. Thermodynamic properties of the heme-binding domain of the O2 sensor, FixL, in the apo and heme-loaded forms were analyzed using UV/vis and far-UV CD spectroscopy, and found that addition of heme increased the amount of structure and stability of the protein. Primary structure analysis of various b-heme proteins showed a trend of intrinsic disorder in the heme pocket. These proteins were further analyzed using the COREX program to determine if structural properties of an apoprotein could be revealed from the holoprotein structure. For some proteins, there was a relation between COREX output and known apoprotein properties. How specificity for heme is controlled was studied using binding experiments of a chlorophyll analog with myoglobin and two cyanobacterial hemoglobins, and monitored by UV/vis spectroscopy. Unlike myoglobin, the chlorophyll derivative was not specifically bound by the hemoglobins. A structural motif proposed to be present in disordered regions under folding conditions is the inverse gamma-turn. A polyalanine peptide, too short for stable secondary structure, was examined under various solvent conditions by far-UV CD and NMR, though no clear evidence of the gamma-turn was found. A recognition site within an IDR has been shown to form an alpha-helix when bound to enolase. Peptides modeled after this site were analyzed by far-UV CD and NMR, and found to maintain no secondary structure in solution, but structure was induced by addition of TFE. In general, a variety of systems were utilized to better understand the requirements for polypeptides to fold. |
