Amelia A. Fuller

An Experimental Survey of the Transition Between Two-State and Downhill Protein Folding Scenarios.

A kinetic and thermodynamic survey of 35 WW domain sequences is used in combination with a model to discern the energetic requirements for the transition from two-state folding to downhill folding. The sequences used exhibit a 600-fold range of folding rates at the temperature of maximum folding rate. Very stable proteins can achieve complete downhill folding when the temperature is lowered sufficiently below the melting temperature, and then at even lower temperatures they become two-state folders again because of cold denaturation. Less stable proteins never achieve a sufficient bias to fold downhill because of the onset of cold denaturation. The model, considering both heat and cold denaturation, reveals that to achieve incipient downhill folding (barrier <3 RT) or downhill folding (no barrier), the WW domain average melting temperatures have to be ≥50°C for incipient downhill folding and ≥90°C for downhill folding.

A cross-strand Trp Trp pair stabilizes the hPin1 WW domain at the expense of function.

Using the human Pin1 WW domain (hPin1 WW), we show that replacement of two nearest neighbor non‐hydrogen‐bonded residues on adjacent β‐strands with tryptophan (Trp) residues increases β‐sheet thermodynamic stability by 4.8 kJ mol−1 at physiological temperature. One‐dimensional NMR studies confirmed that introduction of the Trp–Trp pair does not globally perturb the structure of the triple‐stranded β‐sheet, while circular dichroism studies suggest that the engineered cross‐strand Trp–Trp pair adopts a side‐chain conformation similar to that first reported for a designed “Trp‐zipper” β‐hairpin peptide, wherein the indole side chains stack perpendicular to each other. Even though the mutated side chains in wild‐type hPin1 WW are not conserved among WW domains and compose the β‐sheet surface opposite to that responsible for ligand binding, introduction of the cross‐strand Trp–Trp pair effectively eliminates hPin1 WW function as assessed by the loss of binding affinity toward a natural peptide ligand. Maximizing both thermodynamic stability and the domain function of hPin1 WW by the above mentioned approach appears to be difficult, analogous to the situation with loop 1 optimization explored previously. That introduction of a non‐hydrogen‐bonded cross‐strand Trp–Trp pair within the hPin1 WW domain eliminates function may provide a rationale for why this energetically favorable pairwise interaction has not yet been identified in WW domains or any other biologically evolved protein with known three‐dimensional structure.

Self-Association of Water-Soluble Peptoids Comprising (S)-N-1-(Naphthylethyl)glycine Residues

Peptoids (N-substituted glycine oligomers) are widely used peptidomimetics, and an enhanced understanding of their structures is needed to expand their utility, particularly in aqueous applications. We report the synthesis and structural study of four water-soluble peptoids that include strongly helix-promoting (S)-N-1-(naphthylethyl)glycine residues. Peptoid structure changes with both peptoid length and solvent composition. Multiple data support the self-association of the longest peptoid studied here, 1, via hydrophobic interactions in aqueous solutions.

Evaluating β-turn mimics as β-sheet folding nucleators

β-Turns are common conformations that enable proteins to adopt globular structures, and their formation is often rate limiting for folding. β-Turn mimics, molecules that replace the i + 1 and i + 2 amino acid residues of a β-turn, are envisioned to act as folding nucleators by preorganizing the pendant polypeptide chains, thereby lowering the activation barrier for β-sheet formation. However, the crucial kinetic experiments to demonstrate that β-turn mimics can act as strong nucleators in the context of a cooperatively folding protein have not been reported. We have incorporated 6 β-turn mimics simulating varied β-turn types in place of 2 residues in an engineered β-turn 1 or β-bulge turn 1 of the Pin 1 WW domain, a three-stranded β-sheet protein. We present 2 lines of kinetic evidence that the inclusion of β-turn mimics alters β-sheet folding rates, enabling us to classify β-turn mimics into 3 categories: those that are weak nucleators but permit Pin WW folding, native-like nucleators, and strong nucleators. Strong nucleators accelerate folding relative to WW domains incorporating all α-amino acid sequences. A solution NMR structure reveals that the native Pin WW β-sheet structure is retained upon incorporating a strong E-olefin nucleator. These β-turn mimics can now be used to interrogate protein folding transition state structures and the 2 kinetic analyses presented can be used to assess the nucleation capacity of other β-turn mimics.

Evaluating beta-turn mimics as beta-sheet folding nucleators.

β-Turns are common conformations that enable proteins to adopt globular structures, and their formation is often rate limiting for folding. β-Turn mimics, molecules that replace the i + 1 and i + 2 amino acid residues of a β-turn, are envisioned to act as folding nucleators by preorganizing the pendant polypeptide chains, thereby lowering the activation barrier for β-sheet formation. However, the crucial kinetic experiments to demonstrate that β-turn mimics can act as strong nucleators in the context of a cooperatively folding protein have not been reported. We have incorporated 6 β-turn mimics simulating varied β-turn types in place of 2 residues in an engineered β-turn 1 or β-bulge turn 1 of the Pin 1 WW domain, a three-stranded β-sheet protein. We present 2 lines of kinetic evidence that the inclusion of β-turn mimics alters β-sheet folding rates, enabling us to classify β-turn mimics into 3 categories: those that are weak nucleators but permit Pin WW folding, native-like nucleators, and strong nucleators. Strong nucleators accelerate folding relative to WW domains incorporating all α-amino acid sequences. A solution NMR structure reveals that the native Pin WW β-sheet structure is retained upon incorporating a strong E-olefin nucleator. These β-turn mimics can now be used to interrogate protein folding transition state structures and the 2 kinetic analyses presented can be used to assess the nucleation capacity of other β-turn mimics.

A fluorescent peptoid pH-sensor

Peptoids, N-substituted glycine oligomers, can adopt stable three-dimensional structures and have found diverse application as peptide surrogates and as nanomaterials. In this report, we have expanded peptoid function to include pH sensing by coupling pH-induced peptoid conformational changes with fluorescence intensity changes. We report two new peptoids (2 and 3) that comprise carboxylic-acid functionalized side chains and undergo conformational rearrangement in response to pH. Peptoids 2 and 3 are also labeled at one side-chain with an environmentally sensitive fluorophore, 4-N,N-dimethylamino-1,8-naphthalimide (4DMN). The fluorescence intensity of 2 varies 24-fold over the pH range studied. These spectroscopic properties make 2 a sensitive, biocompatible pH sensor.

A FlAsH-tetracysteine assay for quantifying the association and orientation of transmembrane α-helices.

The association of membrane proteins with a single transmembrane a-helix (TMH) has been shown to play a critical role in numerous cellular processes. For example, TMH dimerization of the ErbB family of receptor tyrosine kinases (RTKs) results in trans-phosphorylation and subsequent activation of the downstream pathways of growth factor signalling. A series of naturally occurring, single amino acid mutations in the TMH domain of these RTKs have been linked to a number of diseases, presumably because these mutations perturb TMH dimerization propensities. For example, the dimer promoting mutation V664E in the TMH domain of ErbB2 (HER2) has been associated with an increased risk of cancer. This physiological importance is reflected in the high number of drugs that target membrane proteins (ca. 50%) relative to their distribution in the proteome (ca. 30%). Although significant progress has been made in our mechanistic understanding of protein association in membranes, it remains difficult to predict the oligomerization state of a TMH and rationalize the (patho)physiological consequences of a mutation within a membrane protein. While the association of soluble proteins is primarily driven by the hydrophobic effect, protein–protein interaction within membranes is thought to rely on weak molecular interactions such as sidechain hydrogen bonding and van der Waal’s packing. In order to gain a better understanding of these energetic factors and ultimately develop novel inhibitors of TMH association, a simple and efficient assay is highly desirable for quantifying TMH association both in vitro (detergent micelles and liposomes) and in living cells. Biarsenical dyes, such as 4’,5’bis(1,3,2-dithioarsolan-2-yl)fluorescein (FlAsH), are an intriguing tool for monitoring protein–protein interaction in membranes. In their EDT (ethane-1,2-dithiol)-protected forms, these dyes (e.g. , FlAsH–EDT2) are essentially nonfluorescent. However, thiol–arsenic ligand exchange with a tC motif elicits a strong fluorescence emission. These fluorophores have been applied to a number of soluble proteins to report their folding and stability, and even subtle conformational changes. A recent development in this area has resulted in a technique known as “bipartite tC display”, in which the biarsenical dyes are used to monitor the dimerization of water-soluble proteins. Herein, we describe the development of a FlAsH–tC assay for quantifying the association of membrane-embedded proteins. While the commonly used methods, such as SDSPAGE and analytical ultracentrifugation (AUC) are limited to micellar systems, the FlAsH–tC assay allows facile evaluation of protein dimerization in both micelles and lipid bilayers. Importantly, the FlAsH fluorescence reports TMH dimerization in an orientation-specific manner (vide infra), which is advantageous over the previously reported, FRET-based assays. As a model system, we first synthesised a pair of polyleucine (pL)-based peptides (Figure 1B). These peptides were chosen because their oligomerization state had been previously characterized in both micelles (by AUC and SDS-PAGE) and the

Evaluating -turn mimics as -sheet folding nucleators

β-Turns are common conformations that enable proteins to adopt globular structures, and their formation is often rate limiting for folding. β-Turn mimics, molecules that replace the i + 1 and i + 2 amino acid residues of a β-turn, are envisioned to act as folding nucleators by preorganizing the pendant polypeptide chains, thereby lowering the activation barrier for β-sheet formation. However, the crucial kinetic experiments to demonstrate that β-turn mimics can act as strong nucleators in the context of a cooperatively folding protein have not been reported. We have incorporated 6 β-turn mimics simulating varied β-turn types in place of 2 residues in an engineered β-turn 1 or β-bulge turn 1 of the Pin 1 WW domain, a three-stranded β-sheet protein. We present 2 lines of kinetic evidence that the inclusion of β-turn mimics alters β-sheet folding rates, enabling us to classify β-turn mimics into 3 categories: those that are weak nucleators but permit Pin WW folding, native-like nucleators, and strong nucleators. Strong nucleators accelerate folding relative to WW domains incorporating all α-amino acid sequences. A solution NMR structure reveals that the native Pin WW β-sheet structure is retained upon incorporating a strong E-olefin nucleator. These β-turn mimics can now be used to interrogate protein folding transition state structures and the 2 kinetic analyses presented can be used to assess the nucleation capacity of other β-turn mimics.

Research into selective biomarkers of erythrocyte exposure to organophosphorus compounds.

Flowcytometric procedures provide distinct advantages over the colorimetric methods currently in use to monitor erythrocytes for exposure of patients to organophosphorus (OP) pesticides and chemical warfare agents; therefore, they warrant exploration. Two types of fluorescent probes-one to detect the total acetylcholinesterase on erythrocytes (RBC-AChE) and the other to distinguish between the active and OP-inhibited RBC-AChE-have been explored. Our studies demonstrate that a fluorescently conjugated fasciculin can be used to monitor total, active, and OP-inhibited RBC-AChE. However, a fluorescently tagged potent inhibitor of AChE, TZ2PIQ-A6 with a K(d) of 33 fM, did not distinguish between the active and OP-inhibited RBC-AChE, nor did three different biotinylated OP compounds. The biotin-fluorescent avidin approach is not a viable procedure for monitoring RBC-AChE. Western blot studies indicate that there are at least 20 serine hydrolases on the surface of red blood cells (RBCs). Plans currently under way for the development of more specific probes to distinguish between active and OP-inhibited RBC-AChE are discussed.

A peptoid supramolecular host for benzo[a]pyrene in water

Abstract Studies that identify a new supramolecular host for the highly toxic and prevalent carcinogen benzo[a]pyrene (BP) are reported here. By monitoring changes to absorption and fluorescence spectra, the interaction between BP and two peptoids (N-substituted glycine oligomers) of different length and self-association propensities were compared. Peptoid length and solvent choice influence spectral features. Observed trends support that peptoid self-association in aqueous solution generates a hydrophobic core to which BP can associate.