Biomedical subjects

Athi N Naganathan

Publications and source records attributed to Athi N Naganathan.

4 recordsLinked to original sources

PubMed2006

Dynamics, energetics, and structure in protein folding.

For many decades, protein folding experimentalists have worked with no information about the time scales of relevant protein folding motions and without methods for estimating the height of folding barriers. Protein folding experiments have been interpreted using chemical models in which the folding process is characterized as a series of equilibria between two or more distinct states that interconvert with activated kinetics. Accordingly, the information to be extracted from experiments was circumscribed to apparent equilibrium constants and relative folding rates. Recent developments are changing this situation dramatically. The combination of fast-folding experiments with the development of analytical methods more closely connected to physical theory reveals that folding barriers in native conditions range from minimally high (approximately 14RT for the very slow folder AcP) to nonexistent. While slow-folding (i.e., > or = 1 ms) single-domain proteins are expected to fold in a two-state fashion, microsecond-folding proteins should exhibit complex behavior arising from crossing marginal or negligible folding barriers. This realization opens a realm of exciting opportunities for experimentalists. The free energy surface of a protein with a marginal (or no) barrier can be mapped using equilibrium experiments, which could resolve energetic factors from structural factors in folding. Kinetic experiments on these proteins provide the unique opportunity to measure folding dynamics directly. Furthermore, the complex distributions of time-dependent folding behaviors expected for these proteins might be accessible to single-molecule measurements. Here, we discuss some of these recent developments in protein folding, emphasizing aspects that can serve as a guide for experimentalists interested in exploiting this new avenue of research.

Entropy↗

PubMed2005

Direct measurement of barrier heights in protein folding.

A serious limitation in the study of protein folding reactions resides in the lack of experimental methods to measure the absolute height of the free energy barrier. This is particularly unfortunate given that if folding barriers are small, as theory predicts, it might be possible to resolve folding mechanisms directly. Here we explore the performance of a recently developed method to extract folding barriers from equilibrium differential scanning calorimetry (DSC) experiments. To test the method, we compare the thermodynamic barrier heights for 15 proteins obtained from available DSC data with the folding rates measured in kinetic experiments. The correlation between these two parameters is very good (r2 = 0.9) and has a slope consistent with the same energy scale. These results confirm that it is possible to measure free energy barriers for natural proteins from equilibrium DSC experiments. Furthermore, the measured barrier heights are small (<8 RT), in general, and marginal or nonexisting for fast-folding proteins.

Calorimetry, Differential Scanning↗

PubMed2005

Robustness of downhill folding: guidelines for the analysis of equilibrium folding experiments on small proteins.

Previously, we identified the protein BBL as a downhill folder. This conclusion was based on the statistical mechanical analysis of equilibrium experiments performed in two variants of BBL, one with a fluorescent label at the N-terminus, and another one labeled at both ends. A recent report has claimed that our results are an artifact of label-induced aggregation and that BBL with no fluorescent labels and a longer N-terminal tail folds in a two-state fashion. Here, we show that singly and doubly labeled BBL do not aggregate, unfold reversibly, and have the same thermodynamic properties when studied under appropriate experimental conditions (e.g., our original conditions (1)). With an elementary analysis of the available data on the nonlabeled BBL (2), we also show that this slightly more stable BBL variant is not a two-state folder. We discuss the problems that led to its previous misclassification and how they can be avoided. Finally, we investigate the equilibrium unfolding of the singly labeled BBL with both ends protected by acetylation and amidation. This variant has the same thermodynamic stability of the nonlabeled BBL and displays all the equilibrium signatures of downhill folding. From all these observations, we conclude that fluorescent labels do not perturb the thermodynamic properties of BBL, which consistently folds downhill regardless of its stability and specific protein tails. The work on BBL illustrates the shortcomings of applying conventional procedures intended to distinguish between two-state and three-state folding models to small fast-folding proteins.

Amino Acid Sequence↗

PubMed2005

Scaling of folding times with protein size.

Current experimental data show a 9-orders-of-magnitude span in the folding times of proteins. Such a wide range is typically considered a direct consequence of the complexity in structural and sequence patterns of natural proteins. By using a database of 69 proteins and peptides analyzed experimentally, we observe that the folding time scales with the number of residues in the protein. The correlation coefficient is 0.74 or higher, and indicates that it is possible to predict the folding time of a protein with a precision of approximately 1.1 times decades from just its size. A simple thermodynamic analysis of this correlation suggests that the smallest proteins are expected to have very marginal free energy barriers to folding.

DNA-Binding Proteins↗