SignificanceEfficient synthesis and folding of proteins, avoiding misfolded states, are central to cell function. As folding may be initiated in parallel with translation, key experimental challenges are to map changes that occur in folding free energy landscapes as translation proceeds and to understand how these landscapes might be modulated by the ribosome and auxiliary factors. Here, we study the length-dependent folding of a domain from a tandem repeat protein and solve the structure of a stable folding intermediate. Although destabilized by the ribosome at equilibrium, modeling of the nonequilibrium folding pathway nevertheless indicates a significant role for proline isomerization during translation. We develop a simple model to explore the impact of cotranslational folding kinetics on misfolding hazards.

2D-IR spectroscopy of site-specifically incorporated –SCN labels in the calcium sensor protein calmodulin reveals local dynamics and solvation as they change upon calcium uptake and protein–ligand interaction.

Cysteine S−H bonds have a spectroscopically convenient stretching frequency of ~2550 cm−1. Their cross-section, however, is low, and the band can be strongly broadened in heterogeneous environments, making detection very challenging. With two-dimensional infrared (2D-IR) setups achieving ever higher sensitivities in recent years, systematic use of the weak Cys−SH absorption band is now within reach, even at low millimolar protein concentrations. Here we demonstrate the capabilities of Cys−SH as an intrinsic 2D-IR label in pyruvate oxidase from E. coli ( EcPOX), an enzyme with ten cysteines in its native sequence. 1D-IR measurements on the wild-type and individual cysteine knock-out variants show that two such residues have especially narrow SH signatures, caused by their intrahelical hydrogen bonding. 2D-IR analysis of these bands reveals an extraordinarily high anharmonicity (~110 cm−1) and a long vibrational lifetime (~4 ps). This allows monitoring spectral diffusion via center line slope analysis for up to 10 ps-separately for both, the ground and excited state. The unique spectroscopic features and its ease of introduction make Cys−SH a useful IR spectroscopic label.