Effects of maturation on the conformational free-energy landscape of SOD1.

Amyotrophic lateral sclerosis (ALS) is a devastating fatal syndrome characterized by very rapid degeneration of motor neurons. A leading hypothesis is that ALS is caused by toxic protein misfolding and aggregation, as also occurs in many other neurodegenerative disorders, such as prion, Alzheimer's, Parkinson's, and Huntington's diseases. A prominent cause of familial ALS is mutations in the protein superoxide dismutase (SOD1), which promote the formation of misfolded SOD1 conformers that are prone to aberrant interactions both with each other and with other cellular components. We have shown previously that immature SOD1, lacking bound Cu and Zn metal ions and the intrasubunit disulfide bond (apoSOD12SH), has a rugged free-energy surface (FES) and exchanges with four other conformations (excited states) that have millisecond lifetimes and sparse populations on the order of a few percent. Here, we examine further states of SOD1 along its maturation pathway, as well as those off-pathway resulting from metal loss that have been observed in proteinaceous inclusions. Metallation and disulfide bond formation lead to structural transformations including local ordering of the electrostatic loop and native dimerization that are observed in rare conformers of apoSOD12SH; thus, SOD1 maturation may occur via a population-switch mechanism whereby posttranslational modifications select for preexisting structures on the FES. Metallation and oxidation of SOD1 stabilize the native, mature conformation and decrease the number of detected excited conformational states, suggesting that it is the immature forms of the protein that contribute to misfolded conformations in vivo rather than the highly stable enzymatically active dimer.

Tuning the Attempt Frequency of Protein Folding Dynamics via Transition-State Rigidification: Application to Trp-Cage.

The attempt frequency or prefactor (k0) of the transition-state rate equation of protein folding kinetics has been estimated to be on the order of 10(6) s(-1), which is many orders of magnitude smaller than that of chemical reactions. Herein we use the mini-protein Trp-cage to show that it is possible to significantly increase the value of k0 for a protein folding reaction by rigidifying the transition state. This is achieved by reducing the conformational flexibility of a key structural element (i.e., an α-helix) formed in the transition state via photoisomerization of an azobenzene cross-linker. We find that this strategy not only decreases the folding time of the Trp-cage peptide by more than an order of magnitude (to ∼100 ns at 25°C) but also exposes parallel folding pathways, allowing us to provide, to the best of our knowledge, the first quantitative assessment of the curvature of the transition-state free-energy surface of a protein.

Site-specific infrared probes of proteins.

Infrared spectroscopy has played an instrumental role in the study of a wide variety of biological questions. However, in many cases, it is impossible or difficult to rely on the intrinsic vibrational modes of biological molecules of interest, such as proteins, to reveal structural and environmental information in a site-specific manner. To overcome this limitation, investigators have dedicated many recent efforts to the development and application of various extrinsic vibrational probes that can be incorporated into biological molecules and used to site-specifically interrogate their structural or environmental properties. In this review, we highlight recent advancements in this rapidly growing research area.

Experimental validation of the role of trifluoroethanol as a nanocrowder.

Trifluoroethanol (TFE) is commonly used to induce protein secondary structure, especially α-helix formation. Due to its amphiphilic nature, however, TFE can also self-associate to form micellelike, nanometer-sized clusters. Herein, we hypothesize that such clusters can act as nanocrowders to increase protein folding rates via the excluded volume effect. To test this hypothesis, we measure the conformational relaxation kinetics of an intrinsically disordered protein, the phosphorylated kinase inducible domain (pKID), which forms a helix-turn-helix in TFE solutions. We find that the conformational relaxation rate of pKID displays a rather complex dependence on TFE percentage (v/v): while it first decreases between 0 and 5%, between 5 and 15% the rate increases and then remains relatively unchanged between 15 and 30% and finally decreases again at higher percentages (i.e., 50%). This trend coincides with the fact that TFE clustering is maximized in the range of 15-30%, thus providing validation of our hypothesis. Another line of supporting evidence comes from the observation that the relaxation rate of a monomeric helical peptide, which due to its predominantly local interactions in the folded state is less affected by crowding, does not show a similar TFE dependence.

How quickly can a ß-hairpin fold from its transition state?

Understanding the structural nature of the free energy bottleneck(s) encountered in protein folding is essential to elucidating the underlying dynamics and mechanism. For this reason, several techniques, including Φ-value analysis, have previously been developed to infer the structural characteristics of such high free-energy or transition states. Herein we propose that one (or few) appropriately placed backbone and/or side chain cross-linkers, such as disulfides, could be used to populate a thermodynamically accessible conformational state that mimics the folding transition state. Specifically, we test this hypothesis on a model ß-hairpin, Trpzip4, as its folding mechanism has been extensively studied and is well understood. Our results show that cross-linking the two ß-strands near the turn region increases the folding rate by an order of magnitude, to about (500 ns)(−1), whereas cross-linking the termini results in a hyperstable ß-hairpin that has essentially the same folding rate as the uncross-linked peptide. Taken together, these findings suggest that cross-linking is not only a useful strategy to manipulate folding free energy barriers, as shown in other studies, but also, in some cases, it can be used to stabilize a folding transition state analogue and allow for direct assessment of the folding process on the downhill side of the free energy barrier. The calculated free energy landscape of the cross-linked Trpzip4 also supports this picture. An empirical analysis further suggests, when folding of ß-hairpins does not involve a significant free energy barrier, the folding time (τ) follows a power law dependence on the number of hydrogen bonds to be formed (n(H)), namely, τ = τ(0)n(H)(α), with τ(0) = 20 ns and α = 2.3.

Tightening up the structure, lighting up the pathway: Application of molecular constraints and light to manipulate protein folding, self-assembly and function.

Chemical cross-linking provides an effective avenue to reduce the conformational entropy of polypeptide chains and hence has become a popular method to induce or force structural formation in peptides and proteins. Recently, other types of molecular constraints, especially photoresponsive linkers and functional groups, have also found increased use in a wide variety of applications. Herein, we provide a concise review of using various forms of molecular strategies to constrain proteins, thereby stabilizing their native states, gaining insight into their folding mechanisms, and/or providing a handle to trigger a conformational process of interest with light. The applications discussed here cover a wide range of topics, ranging from delineating the details of the protein folding energy landscape to controlling protein assembly and function.

Using D-Amino Acids to Delineate the Mechanism of Protein Folding: Application to Trp-cage.

Using the miniprotein Trp-cage as a model, we show that D-amino acids can be used to facilitate the delineation of protein folding mechanism. Specifically, we study the folding-unfolding kinetics of three Trp-cage mutants where the native glycine residue near the C-terminus of the α-helix is replaced by a D-amino acid. A previous study showed that these mutations increase the Trp-cage stability, due to a terminal capping effect. Our results show that the stabilizing effect of D-asparagine and D-glutamine originates almost exclusively from a decrease in the unfolding rate, while the D-alanine mutation results in a similar decrease in the unfolding rate, but it also increases the folding rate. Together, these results support a folding mechanism wherein the α-helix formation in the transition state is nucleated at the N-terminus, whereas those long-range native interactions stabilizing this helix are developed at the downhill side of the folding free energy barrier.

Assessment of local friction in protein folding dynamics using a helix cross-linker.

Internal friction arising from local steric hindrance and/or the excluded volume effect plays an important role in controlling not only the dynamics of protein folding but also conformational transitions occurring within the native state potential well. However, experimental assessment of such local friction is difficult because it does not manifest itself as an independent experimental observable. Herein, we demonstrate, using the miniprotein trp-cage as a testbed, that it is possible to selectively increase the local mass density in a protein and hence the magnitude of local friction, thus making its effect directly measurable via folding kinetic studies. Specifically, we show that when a helix cross-linker, m-xylene, is placed near the most congested region of the trp-cage it leads to a significant decrease in both the folding rate (by a factor of 3.8) and unfolding rate (by a factor of 2.5 at 35 °C) but has little effect on protein stability. Thus, these results, in conjunction with those obtained with another cross-linked trp-cage and two uncross-linked variants, demonstrate the feasibility of using a nonperturbing cross-linker to help quantify the effect of internal friction. In addition, we estimate that a m-xylene cross-linker could lead to an increase in the roughness of the folding energy landscape by as much as 0.4-1.0k(B)T.

Using VIPT-jump to distinguish between different folding mechanisms: application to BBL and a Trpzip.

Protein folding involves a large number of sequential molecular steps or conformational substates. Thus, experimental characterization of the underlying folding energy landscape for any given protein is difficult. Herein, we present a new method that can be used to determine the major characteristics of the folding energy landscape in question, e.g., to distinguish between activated and barrierless downhill folding scenarios. This method is based on the idea that the conformational relaxation kinetics of different folding mechanisms at a given final condition will show different dependences on the initial condition. We show, using both simulation and experiment, that it is possible to differentiate between disparate kinetic folding models by comparing temperature jump (T-jump) relaxation traces obtained with a fixed final temperature and varied initial temperatures, which effectively varies the initial potential (VIP) of the system of interest. We apply this method (hereafter refer to as VIPT-jump) to two model systems, tryptophan zipper (Trpzip)-2c and BBL, and our results show that BBL exhibits characteristics of barrierless downhill folding, whereas Trpzip-2c folding encounters a free energy barrier. In addition, using the T-jump data of BBL we are able to provide, via Langevin dynamics simulations, a realistic estimate of its conformational diffusion coefficient.

Using thioamides to site-specifically interrogate the dynamics of hydrogen bond formation in ß-sheet folding.

Thioamides are sterically almost identical to their oxoamide counterparts, but they are weaker hydrogen bond acceptors. Therefore, thioamide amino acids are excellent candidates for perturbing the energetics of backbone-backbone H-bonds in proteins and hence should be useful in elucidating protein folding mechanisms in a site-specific manner. Herein, we validate this approach by applying it to probe the dynamic role of interstrand H-bond formation in the folding kinetics of a well-studied ß-hairpin, tryptophan zipper. Our results show that reducing the strength of the peptide's backbone-backbone H-bonds, except the one directly next to the ß-turn, does not change the folding rate, suggesting that most native interstrand H-bonds in ß-hairpins are formed only after the folding transition state.

Site-Specific Spectroscopic Reporters of the Local Electric Field, Hydration, Structure, and Dynamics of Biomolecules.

Elucidating the underlying molecular mechanisms of protein folding and function is a very exciting and active research area, but poses significant challenges. This is due in part to the fact that existing experimental techniques are incapable of capturing snapshots along the 'reaction coordinate' in question with both sufficient spatial and temporal resolutions. In this regard, recent years have seen increased interests and efforts in development and employment of site-specific probes to enhance the structural sensitivity of spectroscopic techniques in conformational and dynamical studies of biological molecules. In particular, the spectroscopic and chemical properties of nitriles, thiocyanates, and azides render these groups attractive for the interrogation of complex biochemical constructs and processes. Here, we review their signatures in vibrational, fluorescence and NMR spectra and their utility in the context of elucidating chemical structure and dynamics of protein and DNA molecules.

Selective incorporation of nitrile-based infrared probes into proteins via cysteine alkylation.

The nitrile stretching vibration is increasingly used as a sensitive infrared probe of local protein environments. However, site-specific incorporation of a nitrile moiety into proteins is difficult. Here we show that various aromatic nitriles can be easily incorporated into peptides and proteins via either thiol alkylation or arylation reaction.