Michelle R. Bunagan

Achieving Secondary Structural Resolution in Kinetic Measurements of Protein Folding: A Case Study of the Folding Mechanism of Trp-cage

Protein folding kinetics are often measured by monitoring the change of a single spectroscopic signal, such as the fluorescence of an intrinsic fluorophore or the absorbance at a single frequency within an electronic or vibrational band of the protein backbone. While such an experimental strategy is easy to implement, the use of a single spectroscopic signal can leave important folding events undetected and overlooked. Herein, we demonstrate, using the mini-protein Trp-cage as an example, that the structural resolution of protein folding kinetics can be significantly improved when a multi-probe and multi-frequency approach is used, thus allowing a more complete understanding of the folding mechanism.

Folding Kinetics of a Naturally Occurring Helical Peptide: Implication of the Folding Speed Limit of Helical Proteins

The folding mechanism and dynamics of a helical protein may strongly depend on how quickly its constituent alpha-helices can fold independently. Thus, our understanding of the protein folding problem may be greatly enhanced by a systematic survey of the folding rates of individual alpha-helical segments derived from their parent proteins. As a first step, we have studied the relaxation kinetics of the central helix (L9:41-74) of the ribosomal protein L9 from the bacterium Bacillus stearothermophilus , in response to a temperature-jump ( T-jump) using infrared spectroscopy. L9:41-74 has been shown to exhibit unusually high helicity in aqueous solution due to a series of side chain-side chain interactions, most of which are electrostatic in nature, while still remaining monomeric over a wide concentration range. Thus, this peptide represents an excellent model system not only for examining how the folding rate of naturally occurring helices differs from that of the widely studied alanine-based peptides, but also for estimating the folding speed limit of (small) helical proteins. Our results show that the T-jump induced relaxation rate of L9:41-74 is significantly slower than that of alanine-based peptides. For example, at 11 degrees C its relaxation time constant is about 2 micros, roughly seven times slower than that of SPE(5), an alanine-rich peptide of similar chain length. In addition, our results show that the folding rate of a truncated version of L9:41-74 is even slower. Taken together, these results suggest that individual alpha-helical segments in proteins may fold on a time scale that is significantly slower than the folding time of alanine-based peptides. Furthermore, we argue that the relaxation rate of L9:41-74 measured between 8 and 45 degrees C provides a realistic estimate of the ultimate folding rate of (small) helical proteins over this temperature range.

Probing the Kinetic Cooperativity of β-Sheet Folding Perpendicular to the Strand Direction†

In an attempt to determine how the folding dynamics of multistranded beta-sheets vary with the strand number, we have studied the temperature-induced relaxation kinetics of a four-stranded beta-sheet, DPDPDP. Our results show that the thermally induced relaxation of DPDPDP occurs on the nanosecond time scale; however, a comparison of the current results with those obtained on a sequence-related, three-stranded beta-sheet suggests that increasing the strand number from three to four increases the folding free energy barrier by a minimum of 0.8 kcal/mol, depending on the folding mechanism. Therefore, these results together suggest that the relaxation kinetics of DPDPDP can be analyzed according to a two-state model even though its folding may actually involve parallel (but degenerate or nearly degenerate) kinetic pathways. The apparent, two-state folding time of DPDPDP is determined to be approximately 0.44 micros at the thermal melting temperature, which makes it one of the fastest folders known to date.

Probing the folding intermediate of Rd-apocyt b562 by protein engineering and infrared T-jump

Small proteins often fold in an apparent two‐state manner with the absence of detectable early‐folding intermediates. Recently, using native‐state hydrogen exchange, intermediates that exist after the rate‐limiting transition state have been identified for several proteins. However, little is known about the folding kinetics from these post‐transition intermediates to their corresponding native states. Herein, we have used protein engineering and a laser‐induced temperature‐jump (T‐jump) technique to investigate this issue and have applied it to Rd‐apocyt b562, a four‐helix bundle protein. Previously, it has been shown that Rd‐apocyt b562 folds via an on‐pathway hidden intermediate, which has only the N‐terminal helix unfolded. In the present study, a double mutation (V16G/I17A) in the N‐terminal helix of Rd‐apocyt b562 was made to further increase the relative population of this intermediate state at high temperature by selectively destabilizing the native state. In the circular dichroism thermal melting experiment, this mutant showed apparent two‐state folding behavior. However, in the T‐jump experiment, two kinetic phases were observed. Therefore, these results are in agreement with the idea that a folding intermediate is populated on the folding pathway of Rd‐apocyt b562. Moreover, it was found that the exponential growth rate of the native state from this intermediate state is roughly (25 μsec)−1 at 65°C.