Local and Large-Scale Conformational Dynamics in Unfolded Proteins and IDPs. I. Effect of Solvent Viscosity and Macromolecular Crowding.

Protein/solvent interactions largely influence protein dynamics, particularly motions in unfolded and intrinsically disordered proteins (IDPs). Here, we apply triplet-triplet energy transfer (TTET) to investigate the coupling of internal protein motions to solvent motions by determining the effect of solvent viscosity (η) and macromolecular crowding on the rate constants of loop formation () in several unfolded polypeptide chains including IDPs. The results show that the viscosity dependence of loop formation depends on amino acid sequence, loop length, and co-solute size.

Local and Large-Scale Conformational Dynamics in Unfolded Proteins and IDPs. II. Effect of Temperature and Internal Friction.

Internal dynamics of proteins are essential for protein folding and function. Dynamics in unfolded proteins are of particular interest since they are the basis for many cellular processes like folding, misfolding, aggregation, and amyloid formation and also determine the properties of intrinsically disordered proteins (IDPs). It is still an open question of what governs motions in unfolded proteins and whether they encounter major energy barriers.

Multimodal Spectroscopic Study of Amyloid Fibril Polymorphism.

Amyloid fibrils are a large class of self-assembled protein aggregates that are formed from unstructured peptides and unfolded proteins. The fibrils are characterized by a universal β-sheet core stabilized by hydrogen bonds, but the molecular structure of the peptide subunits exposed on the fibril surface is variable. Here we show that multimodal spectroscopy using a range of bulk- and surface-sensitive techniques provides a powerful way to dissect variations in the molecular structure of polymorphic amyloid fibrils.

Quantifying Surfactant Alkyl Chain Orientation and Conformational Order from Sum Frequency Generation Spectra of CH Modes at the Surfactant-Water Interface.

We combine second-order nonlinear vibrational spectroscopy and quantum-chemical calculations to quantify the molecular tilt angle and the structural variation of a decanoic acid surfactant monolayer on water. We demonstrate that there is a remarkable degree of delocalization of the vibrational modes along the backbone of the amphiphilic molecule. A simulation-based on modeled sum frequency generation (SFG) spectra offers quantitative insights into the disorder of surfactant monolayers at the water-air interface.

Background-Free Fourth-Order Sum Frequency Generation Spectroscopy.

The recently developed 2D sum frequency generation spectroscopy offers new possibilities to analyze the structure and structural dynamics of interfaces in a surface-specific manner. Its implementation, however, has so far remained limited to the pump-probe geometry, with its inherent restrictions. Here we present 2D SFG experiments utilizing a novel noncollinear geometry of four incident laser pulses generating a 2D SFG response, analogous to the triangle geometry applied in bulk-sensitive 2D infrared spectroscopy.

Nanoscale Heterogeneity of the Molecular Structure of Individual hIAPP Amyloid Fibrils Revealed with Tip-Enhanced Raman Spectroscopy.

Type 2 diabetes mellitus is characterized by the pathological deposition of fibrillized protein, known as amyloids. It is thought that oligomers and/or amyloid fibrils formed from human islet amyloid polypeptide (hIAPP or amylin) cause cell death by membrane damage. The molecular structure of hIAPP amyloid fibrils is dominated by β-sheet structure, as probed with conventional infrared and Raman vibrational spectroscopy.

Model peptides uncover the role of the β-secretase transmembrane sequence in metal ion mediated oligomerization.

The β-secretase or β-site amyloid precursor protein cleaving enzyme 1 (BACE1) is the enzyme responsible for the formation of amyloid-β peptides, which have a major role in Alzheimer pathogenesis. BACE1 has a transmembrane sequence (TMS), which makes it unique among related proteases. We noticed that the BACE1 TMS contains an uncommon sulfur-rich motif.

The polyphenol EGCG inhibits amyloid formation less efficiently at phospholipid interfaces than in bulk solution.

Age-related diseases, like Alzheimer's disease and type 2 diabetes mellitus, are characterized by protein misfolding and the subsequent pathological deposition of fibrillized protein, also called amyloid. Several classes of amyloid-inhibitors have recently been tested, traditionally under bulk conditions. However, it has become apparent that amyloid fibrils and oligomers assemble and exert their cytotoxic effect at cellular membranes, rather than in bulk solution.

Time-resolved methods in biophysics. 10. Time-resolved FT-IR difference spectroscopy and the application to membrane proteins.

The introduction of time-resolved Fourier transform infrared (FT-IR) spectroscopy to biochemistry opened the possibility of monitoring the catalytic mechanism of proteins along their reaction pathways. The infrared approach is very fruitful, particularly in the application to membrane proteins where NMR and X-ray crystallography are challenged by the size and protein hydrophobicity, as well as by their limited time-resolution. Here, we summarize the principles and experimental realizations of time-resolved FT-IR spectroscopy developed in our group and compare with aspects emerging from other laboratories.

Time-resolved flow-flash FT-IR difference spectroscopy: the kinetics of CO photodissociation from myoglobin revisited.

Fourier-transform infrared (FT-IR) difference spectroscopy has been proven to be a significant tool in biospectroscopy. In particular, the step-scan technique monitors structural and electronic changes at time resolutions down to a few nanoseconds retaining the multiplex advantage of FT-IR. For the elucidation of the functional mechanisms of proteins, this technique is currently limited to repetitive systems undergoing a rapid photocycle.