Using synchrotron FTIR, we will examine the secondary structure of lysozyme during the folding process on a sub-millisecond time scale. Lysozyme can be unfolded chemically and refolded by diluting out the denaturant. During folding, this enzyme displays very complex changes in secondary structure with time. With stopped flow UV-CD measurements three kinetic phases were detected. First, the burst phase occurred within the first two milliseconds, at which time the secondary structure, as measured by the UV-CD, reached a level similar to that of the native enzyme. Quite remarkably, the secondary structure continued to develop and at 80 ms reached a level 50% greater than that of the native enzyme! The ellipticity then returned Hto the native value with a time constant of about 300 ms. With the Hinfrared measurements we expect to be able to follow this complete Hprocess but get more information about the specific folding motifs Hfrom the more sensitive infrared technique. The deconvolution of the HAmide I band (1600 - 1700 cm-1), provides details of the percent of Hthe protein structure that is ?-helical, ?-sheet, ?-turn, or extended Hcoil. In our experiments, lysozyme is unfolded with pH and refolded Hby adding salt (KCl). Before examining the folding intermediates in a Htime-resolved fashion with the rapid-mixer (see Subproject 34), first Hwe are determining the equilibrium folding and unfolding conditions. HWe are combining circular dichroism, fluorescence, and infrared Hspectroscopies to confirm the endpoints of the reaction.

National Institute of Health (NIH)
National Center for Research Resources (NCRR)
Biotechnology Resource Grants (P41)
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Albert Einstein College of Medicine
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