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ABSTRACT Chemical exchange line broadening is an important phenomenon in nuclear magnetic resonance (NMR) spectroscopy, in which a nuclear spin experiences more than one magnetic environment as a result of chemical or conformational changes of a molecule. The dynamic process of chemical exchange strongly affects the sensitivity and resolution of NMR experiments and increasingly provides a powerful probe of the interconversion between chemical and conformational states of proteins, nucleic acids, and other biologic macromolecules. A simple and often used theoretic description of chemical exchange in NMR spectroscopy is based on an idealized 2-state jump model (the random phase or telegraph signal). However, chemical exchange can also be represented as a barrier crossing event that can be modeled by using chemical reaction rate theory. The timescale of crossing is determined by the barrier height, the temperature, and the dissipation modeled as collisional or frictional damping. This tutorial explores the connection between the NMR theory of chemical exchange line broadening and strong collision models for chemical kinetics in statistical mechanics. Theoretic modeling and numeric simulation are used to map the rate of barrier crossing dynamics of a particle on a potential energy surface to the chemical exchange relaxation rate constant. By developing explicit models for the exchange dynamics, the tutorial aims to elucidate the underlying dynamical processes that give rise to the rich phenomenology of chemical exchange observed in NMR spectroscopy. Software for generating and analyzing the numeric simulations is provided in the form of Python and Fortran source codes. 

Abstract Protocols for the construction of large, deeply mutagenized protein encoding libraries via Golden Gate assembly of synthetic DNA cassettes employ disparate, system‐specific methodology. Here we present a standardized Golden Gate method for building user‐defined libraries. We demonstrate that a 25 μL reaction, using 40 fmol of input DNA, can generate a library on the order of 1 × 10⁶members and that reaction volume or input DNA concentration can be scaled up with no losses in transformation efficiency. Such libraries can be constructed from dsDNA cassettes generated either by degenerate oligonucleotides or oligo pools. We demonstrate its real‐world effectiveness by building custom, user‐defined libraries on the order of 10⁴–10⁷unique protein encoding variants for two orthogonal protein engineering systems. We include a detailed protocol and provide several general‐use destination vectors.