Residue-specific structural kinetics of proteins through the union of isotope labeling, mid-IR pulse shaping, and coherent 2D IR spectroscopy

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Amyloid formation is implicated in more than 20 human diseases, yet the mechanism by which fibrils form is not well understood. We use 2D infrared spectroscopy and isotope labeling to monitor the kinetics of fibril formation by human islet amyloid polypeptide (hIAPP or amylin) that is associated with type 2 diabetes. We find that an oligomeric intermediate forms during the lag phase with parallel β-sheet structure in a region that is ultimately a partially disordered loop in the fibril. We confirm the presence of this intermediate, using a set of homologous macrocyclic peptides designed to recognize β-sheets. Mutations and molecular dynamics simulations indicate that the intermediate is on pathway. Disrupting the oligomeric β-sheet to form the partially disordered loop of the fibrils creates a free energy barrier that is the origin of the lag phase during aggregation. These results help rationalize a wide range of previous fragment and mutation studies including mutations in other species that prevent the formation of amyloid plaques.inhibitors | aggregation pathway | vibrational coupling T he misfolding of proteins into β-sheet-rich amyloid fibrils is associated with the pathology of more than 20 human diseases, including Alzheimer's, Parkinson, and Huntington diseases (1). Amyloid plaques are formed by masses of fibrils, but growing evidence suggests that the toxic species may be prefibrillar intermediates (2, 3). As a result, there is much interest in understanding the mechanism by which these proteins form fibrils and identifying intermediates in the aggregation pathway. However, obtaining structural information about intermediate species is difficult due to their transient nature. Solid-state NMR (ssNMR) and X-ray crystallography provide high-resolution structures of fibrils (4, 5) and optical techniques can track structural changes in real time (6, 7), but few techniques have both the structural and the temporal resolution to extract specific structural details about intermediates. Fragments have been trapped in intriguing oligomeric structures that may represent intermediate states (5,8) and transient secondary structures are known to exist from circular dichroism measurements and other experiments (9-11), but for full-length proteins it has been difficult to identify the specific residues that contribute to the secondary structure and thus understand their role in the aggregation mechanism. In this paper, we use 2D infrared (2D IR) spectroscopy and isotope labeling to monitor the structural evolution of the full-length human islet amyloid polypeptide (hIAPP or amylin), a 37-residue peptide implicated in type 2 diabetes. We observe the formation of a structured prefibrillar intermediate in a region that has long been known to influence aggregation, but that does not form well-ordered cross-β structure in the amyloid fibril. Its presence provides unique structural insights into the mechanism of amyloid aggregation and helps unify many seemingly inconsistent prior studies.Many studies on hIAPP have focused ...

Section: Methodsmentioning

confidence: 99%

Mechanism of IAPP amyloid fibril formation involves an intermediate with a transient β-sheet

Buchanan

Dunkelberger

Tran

et al. 2013

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345

“…The 2D IR data by themselves cannot distinguish between these two spectral diffusion mechanisms. All of these problems were overcome to obtain the structural dynamics of the functionalized MOF by using 2D IR pulse-shaping techniques (12,16,17) and polarization-selective IR pump-probe experiments.…”

mentioning

confidence: 99%

Structural dynamics inside a functionalized metal–organic framework probed by ultrafast 2D IR spectroscopy

Nishida

Tamimi

Fei

et al. 2014

108

The structural elasticity of metal-organic frameworks (MOFs) is a key property for their functionality. Here, we show that 2D IR spectroscopy with pulse-shaping techniques can probe the ultrafast structural fluctuations of MOFs. 2D IR data, obtained from a vibrational probe attached to the linkers of UiO-66 MOF in low concentration, revealed that the structural fluctuations have time constants of 7 and 670 ps with no solvent. Filling the MOF pores with dimethylformamide (DMF) slows the structural fluctuations by reducing the ability of the MOF to undergo deformations, and the dynamics of the DMF molecules are also greatly restricted. Methodology advances were required to remove the severe light scattering caused by the macroscopic-sized MOF particles, eliminate interfering oscillatory components from the 2D IR data, and address Förster vibrational excitation transfer.2D IR spectroscopy | metal-organic framework | UiO-66 MOF | ultrafast structural fluctuations | solvent confinement effect M etal-organic frameworks (MOFs) are molecular architectures in which metal clusters are connected by organic linkers to yield relatively regular 3D coordination polymers with nanometer-sized pores (1, 2). MOFs have been investigated for a wide variety of chemical applications, such as adsorption of gases (3) and heterogeneous catalysts (4). Among many types of porous materials, MOFs are unique because of their structural elasticity coexisting with a high degree of spatial regularity. In some sense, MOFs are like crystals and polymers. They have relatively regular structures like a crystal, but the metal clusters are joined by organic linkers that, in some systems, produce significant structural mobility. The elasticity of MOFs is intimately related to their physical properties and behavior (5).An important goal for understanding the nature of MOFs and how their chemical composition influences their properties and applications is to develop and apply an experimental method that can measure the ultrafast structural motions of MOFs. The relatively slow motions of the framework occurring in submicrosecond to millisecond scales have been studied by NMR and neutron scattering (6). However, until now, quantitative measurements that can characterize the time dependence of MOF structural motions in ultrafast regimes have not been possible because of the lack of appropriate techniques. Here, we have accomplished the goal by applying ultrafast 2D IR spectroscopy (7) to the study of MOF structural dynamics. 2D IR is akin to 2D NMR, but it operates on the ultrafast timescales necessary to characterize the time dependence of MOF structural motions. In addition, there is the important question of the effects on MOF dynamics when guest molecules fill the MOF nanopores. The guest molecules will affect the structural fluctuations of the framework by interacting with the linkers and the metal units. Furthermore, because of the confinement of guest molecules in MOF nanopores, the dynamics of these molecules are expected to be very different from t...

“…In particular, 2D IR vibrational echo spectroscopy has proven to be useful in gaining insights into proteins' dynamic nature, which is intimately involved in their functions that occur on much longer time scales (11,12). Applications of 2D IR spectroscopy in biology include the study of protein dynamics, structure, folding and unfolding, and enzymatic specificities (13)(14)(15)(16)(17)(18)(19). These studies provide information on the structure and dynamics of proteins and provide a connection between experimental observations and molecular dynamic simulations in the important subnanosecond time regime (20)(21)(22).…”

mentioning

confidence: 99%

Dynamics of the folded and unfolded villin headpiece (HP35) measured with ultrafast 2D IR vibrational echo spectroscopy

Chung

Thielges

Fayer

2011

106

166

A series of two-dimensional infrared vibrational echo experiments performed on nitrile-labeled villin headpiece [HP35-ðCNÞ 2 ] is described. HP35 is a small peptide composed of three alpha helices in the folded configuration. The dynamics of the folded HP35-ðCNÞ 2 are compared to that of the guanidine-induced unfolded peptide, as well as the nitrile-functionalized phenylalanine (PheCN), which is used to differentiate the peptide dynamic contributions to the observables from those of the water solvent. Because the viscosity of solvent has a significant effect on fast dynamics, the viscosity of the solvent is held constant by adding glycerol. For the folded peptide, the addition of glycerol to the water solvent causes observable slowing of the peptide's dynamics. Holding the viscosity constant as GuHCl is added, the dynamics of unfolded peptide are much faster than those of the folded peptide, and they are very similar to that of PheCN. These observations indicate that the local environment of the nitrile in the unfolded peptide resembles that of PheCN, and the dynamics probed by the CN are dominated by the fluctuations of the solvent molecules, in contrast to the observations on the folded peptide.nitrile IR probe | peptide dynamics T he structure of proteins, both folded and unfolded, are inherently dynamic in nature. A protein is constantly undergoing interconversions among a range of structures separated by relatively low energy barriers. Fast conformational sampling can give rise to protein structural evolution on slower time scales. Many experimental and theoretical studies have focused on the native structure due to the relevance of the native state protein to its biological functions, but also because of difficulties associated with characterizing proteins under unfolding conditions, where they can exist in heterogeneous distributions of many conformations (1, 2). Properties of the unfolded state are also significant because they play important roles in folding and stability, transport across membranes, and proteolysis and protein turnover (1). Unfolded proteins have dynamics that are different from the native protein, and the differences in dynamics can shed light on the relationship between the native and the unfolded protein.An attractive target for studying differences between the folded and unfolded structures is the villin headpiece 35 (HP35), a peptide chain composed of 35 amino acids found in a chicken villin as a small subdomain. HP35 folds fast (∼1 μs) compared to large proteins, allowing tractable folding/unfolding simulations. Consequently, it has served as a model system in a number of computational folding studies (3-5), as well as a variety of experimental studies (6-8). NMR and X-ray diffraction studies have indicated that wild-type HP35 folds into three alpha helices (Fig. 1), which encase the hydrophobic core composed of three phenylalanines (9, 10).In this paper, the results of a series of ultrafast 2D IR vibrational echo experiments on nitrile-incorporated HP35 are presented. Two CN-fun...