A simple three-dimensional-focusing, continuous-flow mixer for the study of fast protein dynamics.

We present a simple, yet flexible microfluidic mixer with a demonstrated mixing time as short as 80 μs that is widely accessible because it is made of commercially available parts. To simplify the study of fast protein dynamics, we have developed an inexpensive continuous-flow microfluidic mixer, requiring no specialized equipment or techniques. The mixer uses three-dimensional, hydrodynamic focusing of a protein sample stream by a surrounding sheath solution to achieve rapid diffusional mixing between the sample and sheath.

Differential ordering of the protein backbone and side chains during protein folding revealed by site-specific recombinant infrared probes.

The time scale for ordering of the polypeptide backbone relative to the side chains is a critical issue in protein folding. The interplay between ordering of the backbone and ordering of the side chains is particularly important for the formation of β-sheet structures, as the polypeptide chain searches for the native stabilizing cross-strand interactions. We have studied these issues in the N-terminal domain of protein L9 (NTL9), a model protein with mixed α/β structure.

Implementation of time-resolved step-scan fourier transform infrared (FT-IR) spectroscopy using a kHz repetition rate pump laser.

Time-resolved step-scan Fourier transform infrared (FT-IR) spectroscopy has been shown to be invaluable for studying excited-state structures and dynamics in both biological and inorganic systems. Despite the established utility of this method, technical challenges continue to limit the data quality and more wide ranging applications. A critical problem has been the low laser repetition rate and interferometer stepping rate (both are typically 10 Hz) used for data acquisition.

Ligand trapping by cytochrome c oxidase: implications for gating at the catalytic center.

Cytochrome c oxidase is a member of the heme-copper family of oxygen reductases in which electron transfer is linked to the pumping of protons across the membrane. Neither the redox center(s) associated with proton pumping nor the pumping mechanism presumably common to all heme-copper oxidases has been established. A possible conformational coupling between the catalytic center (Fe(a3)(3+)-Cu(B)(2+)) and a protein site has been identified earlier from ligand binding studies, whereas a structural change initiated by azide binding to the protein has been proposed to facilitate the access of cyanide to the catalytic center of the oxidized bovine enzyme.

Proton interactions with hemes a and a3 in bovine heart cytochrome c oxidase.

Three forms of cytochrome c oxidase, fully oxidized CcO (CcO-O), oxidized CcO complexed with cyanide (CcO.CN), and mixed valence CcO, in which both heme a(3) and Cu(B) are reduced and stabilized by carbon monoxide (MV.CO), were investigated by optical spectroscopy, MCD, and stopped-flow for the pH sensitivity of spectral features.