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.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR001633-18
Application #
6345104
Study Section
Project Start
2000-09-01
Project End
2001-08-31
Budget Start
1998-10-01
Budget End
1999-09-30
Support Year
18
Fiscal Year
2000
Total Cost
Indirect Cost
Name
Albert Einstein College of Medicine
Department
Type
DUNS #
009095365
City
Bronx
State
NY
Country
United States
Zip Code
10461
Vongsvivut, Jitraporn; Fernandez, Jason; Ekgasit, Sanong et al. (2004) Characterization of supported cylinder-planar germanium waveguide sensors with synchrotron infrared radiation. Appl Spectrosc 58:143-51
Masip, Lluis; Pan, Jonathan L; Haldar, Suranjana et al. (2004) An engineered pathway for the formation of protein disulfide bonds. Science 303:1185-9
Huang, Raymond Y; Miller, Lisa M; Carlson, Cathy S et al. (2003) In situ chemistry of osteoporosis revealed by synchrotron infrared microspectroscopy. Bone 33:514-21
Rashidzadeh, Hassan; Khrapunov, Sergei; Chance, Mark R et al. (2003) Solution structure and interdomain interactions of the Saccharomyces cerevisiae ""TATA binding protein"" (TBP) probed by radiolytic protein footprinting. Biochemistry 42:3655-65
Uchida, Takeshi; Takamoto, Keiji; He, Qin et al. (2003) Multiple monovalent ion-dependent pathways for the folding of the L-21 Tetrahymena thermophila ribozyme. J Mol Biol 328:463-78
Taylor, Colleen M; Watton, Stephen P; Bryngelson, Peter A et al. (2003) Inner-sphere complexation of cobalt(II) 2,9-dimethyl-1,10-phenanthroline ([Co(neo)]2+) with commercial and sol-gel derived silica gel surfaces. Inorg Chem 42:312-20
Dewan, John C; Feeling-Taylor, Angela; Puius, Yoram A et al. (2002) Structure of mutant human carbonmonoxyhemoglobin C (betaE6K) at 2.0 A resolution. Acta Crystallogr D Biol Crystallogr 58:2038-42
Kiselar, J G; Maleknia, S D; Sullivan, M et al. (2002) Hydroxyl radical probe of protein surfaces using synchrotron X-ray radiolysis and mass spectrometry. Int J Radiat Biol 78:101-14
Swisher, Jennifer F; Su, Linhui J; Brenowitz, Michael et al. (2002) Productive folding to the native state by a group II intron ribozyme. J Mol Biol 315:297-310
Dhavan, Gauri M; Crothers, Donald M; Chance, Mark R et al. (2002) Concerted binding and bending of DNA by Escherichia coli integration host factor. J Mol Biol 315:1027-37

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