Experimental monitoring and data analysis tools for protein folding: study of steady-state evolution and modeling of kinetic transients by multitechnique and multiexperiment data fusion.
Protein folding is a complex process that can take place through different pathways depending on the inducing agent and on the monitored time scale. This diversity of possibilities requires a good design of experiments and powerful data analysis tools that allow operating with multitechnique measurements and/or with diverse experiments related to different aspects of the process of interest. Multivariate curve resolution-alternating least squares (MCR-ALS) has been the core methodology used to perform multitechnique and/or multiexperiment data analysis. This algorithm allows for obtaining the process concentration profiles and pure spectra of all species involved in the protein folding from the sole raw spectroscopic measurements obtained during the experimental monitoring. The process profiles provide insight on the mechanism of the process studied whereas the shapes of the recovered pure spectra help in the characterization of the protein conformations involved. Relevant features of the MCR-ALS algorithm are the possibility to handle fused data, i.e., series of experiments monitored with different techniques and/or performed under different experimental conditions, and the flexibility to include a priori information linked to general properties of concentration profiles and spectra and to the kinetic model governing the folding process. All these characteristics help to obtain a comprehensive description of the protein folding mechanism. To our knowledge, this work includes for the first time the simultaneous analysis of steady-state and short-time scale kinetic experiments linked to a protein folding process. The potential of this methodology is shown taking myoglobin as a model system for protein folding or, in general, for the study of any complex biological process that needs multitechnique and multiexperiment monitoring and analysis. Transformations in myoglobin due to changes in pH have been monitored by ultraviolet/visible (UV-vis) absorption and circular dichroism (CD) spectroscopy. Steady-state and stopped-flow experiments were carried out to account for the evolution of the process at different time scales. In this example, the multiexperiment analysis has allowed for the reliable detection and modeling of a kinetic transient species in the myoglobin folding process, absent in the steady-state working conditions.
Cutler
Patrick
P
Department of Chemistry, East Carolina University, Greenville, NC 27858, United States.
Gemperline
Paul J
PJ
de Juan
Anna
A
D000818:Animals; D002942:Circular Dichroism; D006736:Horses; D007700:Kinetics; D008954:Models, Biological; D009211:Myoglobin / Q000032:analysis* / Q000378:metabolism*; D017510:Protein Folding
D009211:Myoglobin