G-factor analysis of protein secondary structure in solutions and thin films.
The biological activity of proteins is structure dependent. In this discussion, we describe development of g-factor analysis for characterizing the secondary structure of proteins in solutions and films. In g-factor analysis, experimental circular dichroism (CD) and UV absorbance spectra are converted to dimensionless g-factor spectra by dividing the differential absorbance of circularly polarized light (AL - AR) by the UV absorbance (A) at each wavelength. The spectra can be deconvolved and the secondary structure estimated without information on the protein molecular weight, sample concentration, or sample path length. We have refined g-factor spectral acquisition and deconvolution parameters to improve the precision and accuracy of experimental g-factor spectra and the subsequent secondary structure estimations. In general, slower scan rates and longer response times are required to obtain high quality g-factor spectra than to obtain ordinary CD spectra, particularly for data at wavelengths longer than 230 nm. The spectral acquisition and deconvolution procedures have been validated for aqueous bovine serum albumin (BSA) and poly(L-proline). The secondary structures of fibronectin and laminin in buffer solutions are predicted. When BSA and poly(L-proline) form films on quartz, their secondary structures change significantly: 13% for BSA and 32% for poly(L-proline). By contrast, the secondary structure of fibronectin is the same in solution and films. The g-factor method is an easy, rapid, accurate and precise method for determining secondary structure and structural changes in protein solutions and films. Potential applications range from proteomics and structure-based drug discovery, to the design and fabrication of biosensors, biomaterials and biofluidic devices.
Baker
Brian R
BR
Department of Chemistry and Biochemistry, University of California, Box 951569, Los Angeles, CA 90095-1569, USA.
Garrell
Robin L
RL
D000465:Algorithms; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D005163:Factor Analysis, Statistical; D005353:Fibronectins / Q000737:chemistry; D007797:Laminin / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D010455:Peptides / Q000737:chemistry; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry; D012710:Serum Albumin, Bovine / Q000737:chemistry; D012996:Solutions; D013056:Spectrophotometry, Ultraviolet
D005353:Fibronectins; D007797:Laminin; D010455:Peptides; D011506:Proteins; D012996:Solutions; C011083:polyproline; D012710:Serum Albumin, Bovine