In studies of the ensembles of unfolded structures of a four-helix bundle protein, we have detected the presence of potential precursors of native tertiary structures. These observations were based on the perturbation of NMR chemical shifts of the protein backbone atoms by single site mutations. Some mutations change the chemical shifts of residues remote from the site of mutation indicating the presence of an interaction between the mutated and the remote residues, suggesting that the formation of helix segments and helix-helix interactions is cooperative. We can begin to track down the folding mechanism of this protein using only experimental data by combining the information available for the rate limiting structure formation during the folding process with measurements of the site specific hydrogen bond formation in the burst phase, and with the existence prior to the folding reaction of tertiary structures in the ensemble of otherwise unfolded structures observed in the present study.protein folding | transient structure formation P rotein folding is one of the fundamental processes in living organisms. Genes have been selected in evolution to code for peptide chains with amino acid sequences that fold into well-defined functional structures. Although selection and evolution have ensured that amino acid sequences and the protein folding processes are optimized in any organism, the nature of these mechanisms remains to be fully understood (1).Protein folding has been intensively studied for several decades (2), but so far the atomic details of the folding processes have not been determined experimentally for any protein. The accumulation of detailed knowledge about the mechanisms of protein folding pathways is the tedious and perhaps only way toward understanding the origin of the wealth of protein architectures, which are being formed in the courses of protein folding events. It is relevant, therefore, to develop methods that may describe both the kinetics and the equilibrium thermodynamics of the processes of protein folding. The kinetics of the global protein folding processes are known for many proteins to be very fast, and it has been studied primarily using rapid mixing techniques (3). However, studies of global protein folding processes do not provide any information about those underlying processes, which hold the key to understanding why and how a unique amino acid sequence can form regular protein architecture. Because many proteins do fold spontaneously, there must be an imbedded autonomy in the amino acid sequence directing the course and the result of folding.Here we are concerned with the detection of structure formation in the initial stage of a protein folding event. For this purpose we are using NMR spectroscopy to study the formation of interactions in the unfolded state of acyl coenzyme A binding protein (ACBP) between residues that are separated by more than 10 residues in the amino acid sequence. A number of studies suggest that the transiently populated structures in the unfolded state ...

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Fourier transform infrared (FT-IR) spectroscopy is a valuable technique for characterization of biological samples, providing a detailed fingerprint of the major chemical constituents. However, water vapor and CO(2) in the beam path often cause interferences in the spectra, which can hamper the data analysis and interpretation of results. In this paper we present a new method for removal of the spectral contributions due to atmospheric water and CO(2) from attenuated total reflection (ATR)-FT-IR spectra. In the IR spectrum, four separate wavenumber regions were defined, each containing an absorption band from either water vapor or CO(2). From two calibration data sets, gas model spectra were estimated in each of the four spectral regions, and these model spectra were applied for correction of gas absorptions in two independent test sets (spectra of aqueous solutions and a yeast biofilm (C. albicans) growing on an ATR crystal, respectively). The amounts of the atmospheric gases as expressed by the model spectra were estimated by regression, using second-derivative transformed spectra, and the estimated gas spectra could subsequently be subtracted from the sample spectra. For spectra of the growing yeast biofilm, the gas correction revealed otherwise hidden variations of relevance for modeling the growth dynamics. As the presented method improved the interpretation of the principle component analysis (PCA) models, it has proven to be a valuable tool for filtering atmospheric variation in ATR-FT-IR spectra.