Arnaldo L. Serrano

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.

Spectroscopic studies of protein folding: Linear and nonlinear methods

Although protein folding is a simple outcome of the underlying thermodynamics, arriving at a quantitative and predictive understanding of how proteins fold nevertheless poses huge challenges. Therefore, both advanced experimental and computational methods are continuously being developed and refined to probe and reveal the atomistic details of protein folding dynamics and mechanisms. Herein, we provide a concise review of recent developments in spectroscopic studies of protein folding, with a focus on new triggering and probing methods. In particular, we describe several laser‐based techniques for triggering protein folding/unfolding on the picosecond and/or nanosecond timescales and various linear and nonlinear spectroscopic techniques for interrogating protein conformations, conformational transitions, and dynamics.

Two-Dimensional Sum-Frequency Generation Reveals Structure and Dynamics of a Surface-Bound Peptide

Surface-bound polypeptides and proteins are increasingly used to functionalize inorganic interfaces such as electrodes, but their structural characterization is exceedingly difficult with standard technologies. In this paper, we report the first two-dimensional sum-frequency generation (2D SFG) spectra of a peptide monolayer, which are collected by adding a mid-IR pulse shaper to a standard femtosecond SFG spectrometer. On a gold surface, standard FTIR spectroscopy is inconclusive about the peptide structure because of solvation-induced frequency shifts, but the 2D line shapes, anharmonic shifts, and lifetimes obtained from 2D SFG reveal that the peptide is largely α-helical and upright. Random coil residues are also observed, which do not themselves appear in SFG spectra due to their isotropic structural distribution, but which still absorb infrared light and so can be detected by cross-peaks in 2D SFG spectra. We discuss these results in the context of peptide design. Because of the similar way in which the spectra are collected, these 2D SFG spectra can be directly compared to 2D IR spectra, thereby enabling structural interpretations of surface-bound peptides and biomolecules based on the well-studied structure/2D IR spectra relationships established from soluble proteins.

Instantaneous ion configurations in the K+ ion channel selectivity filter revealed by 2D IR spectroscopy

Potassium channels are responsible for the selective permeation of K+ ions across cell membranes. K+ ions permeate in single file through the selectivity filter, a narrow pore lined by backbone carbonyls that compose four K+ binding sites. Here, we report on the two-dimensional infrared (2D IR) spectra of a semisynthetic KcsA channel with site-specific heavy (13C18O) isotope labels in the selectivity filter. The ultrafast time resolution of 2D IR spectroscopy provides an instantaneous snapshot of the multi-ion configurations and structural distributions that occur spontaneously in the filter. Two elongated features are resolved, revealing the statistical weighting of two structural conformations. The spectra are reproduced by molecular dynamics simulations of structures with water separating two K+ ions in the binding sites, ruling out configurations with ions occupying adjacent sites.

Direct Assessment of the α-Helix Nucleation Time

The nucleation event in α-helix formation is a fundamental process in protein folding. However, determining how quickly it takes place based on measurements of the relaxation dynamics of helical peptides is difficult because such relaxations invariably contain contributions from various structural transitions such as from helical to nonhelical states and helical to partial-helical conformations. Herein, we measure the temperature-jump (T-jump) relaxation kinetics of three model peptides that fold into a single-turn α-helix, using time-resolved infrared spectroscopy, aiming to provide a direct assessment of the helix nucleation rate. The α-helical structure of these peptides is stabilized by a covalent cross-linker formed between the side chains of two residues at the i and i + 4 positions. If we assume that this cross-linker mimics the structural constraint arising from a strong side chain-side chain interaction (e.g., a salt bridge) in proteins, these peptides would represent good models for studying the nucleation process of an α-helix in a protein environment. Indeed, we find that the T-jump induced relaxation rate of these peptides is approximately (0.6 μs)(-1) at room temperature, which is slower than that of commonly studied alanine-based helical peptides but faster than that of a naturally occurring α-helix whose folded state is stabilized by a series of side chain-side chain interactions. Taken together, our results put an upper limit of about 1 μs for the helix nucleation time at 20 °C and suggest that the subsequent propagation steps occur with a time constant of about 240 ns.

Native State Conformational Heterogeneity of HP35 Revealed by Time-Resolved FRET

The villin headpiece subdomain (HP35) has become one of the most widely used model systems in protein folding studies, due to its small size and ultrafast folding kinetics. Here, we use HP35 as a test bed to show that the fluorescence decay kinetics of an unnatural amino acid, p-cyanophenylalanine (Phe(CN)), which are modulated by a nearby quencher (e.g., tryptophan or 7-azatryptophan) through the mechanism of fluorescence resonance energy transfer (FRET), can be used to detect protein conformational heterogeneity. This method is based on the notion that protein conformations having different donor-acceptor distances and interconverting slowly compared to the fluorescence lifetime of the donor (Phe(CN)) would exhibit different donor fluorescence lifetimes. Our results provide strong evidence suggesting that the native free energy basin of HP35 is populated with conformations that differ mostly in the position and mean helicity of the C-terminal helix. This finding is consistent with several previous experimental and computational studies. Moreover, this result holds strong implications for computational investigation of the folding mechanism of HP35.

Mutational analysis of preamyloid intermediates: the role of his-tyr interactions in islet amyloid formation.

Islet amyloid polypeptide (IAPP or Amylin) is a 37-residue, C-terminally amidated pancreatic hormone, cosecreted with insulin that forms islet amyloid in type 2 diabetes. Islet amyloid formation is complex and characterizing preamyloid oligomers is an important topic because oligomeric intermediates are postulated to be the most toxic species produced during fibril formation. A range of competing models for early oligomers have been proposed. The role of the amidated C-terminus in amyloid formation by IAPP and in stabilizing oligomers is not known. Studies with unamidated IAPP have provided evidence for formation of an antiparallel dimer at pH 5.5, stabilized by stacking of His-18 and Tyr-37, but it is not known if this interaction is formed in the physiological form of the peptide. Analysis of a set of variants with a free and with an amidated C-terminus shows that disrupting the putative His-Tyr interaction accelerates amyloid formation, indicating that it is not essential. Amidation to generate the physiologically relevant form of IAPP accelerates amyloid formation, demonstrating that the advantages conferred by C-terminal amidation outweigh increased amyloidogenicity. The analysis of this variant argues that IAPP is not under strong evolutionary pressure to reduce amyloidogenicity. Analysis of an H18Q mutant of IAPP shows that the charge state of the N-terminus is an important factor controlling the rate of amyloid formation, even though the N-terminal region of IAPP is believed to be flexible in the amyloid fibers.

Experimental implementations of 2D IR spectroscopy through a horizontal pulse shaper design and a focal plane array detector

Aided by advances in optical engineering, two-dimensional infrared spectroscopy (2D IR) has developed into a promising method for probing structural dynamics in biophysics and material science. We report two new advances for 2D IR spectrometers. First, we report a fully reflective and totally horizontal pulse shaper, which significantly simplifies alignment. Second, we demonstrate the applicability of mid-IR focal plane arrays (FPAs) as suitable detectors in 2D IR experiments. FPAs have more pixels than conventional linear arrays and can be used to multiplex optical detection. We simultaneously measure the spectra of a reference beam, which improves the signal-to-noise by a factor of 4; and two additional beams that are orthogonally polarized probe pulses for 2D IR anisotropy experiments.

Wide-field FTIR microscopy using mid-IR pulse shaping

We have developed a new table-top technique for collecting wide-field Fourier transform infrared (FTIR) microscopic images by combining a femtosecond pulse shaper with a mid-IR focal plane array. The pulse shaper scans the delay between a pulse pair extremely rapidly for high signal-to-noise, while also enabling phase control of the individual pulses to under-sample the interferograms and subtract background. Infrared absorption images were collected for a mixture of W(CO)₆ or Mn₂(CO)₁₀ absorbed polystyrene beads, demonstrating that this technique can spatially resolve chemically distinct species. The images are sub-diffraction limited, as measured with a USAF test target patterned on CaF₂ and verified with scalar wave simulations. We also find that refractive, rather than reflective, objectives are preferable for imaging with coherent radiation. We discuss this method with respect to conventional FTIR microscopes.