Abstract
The regulated process of protein import into the nucleus of a eukaryotic cell is mediated by specific nuclear localization signals (NLSs) that are recognized by protein import receptors. This study seeks to decipher the energetic details of NLS recognition by the receptor importin alpha through quantitative analysis of variant NLSs. The relative importance of each residue in two monopartite NLS sequences was determined using an alanine scanning approach. These measurements yield an energetic definition of a monopartite NLS sequence where a required lysine residue is followed by two other basic residues in the sequence K(K/R)X(K/R). In addition, the energetic contributions of the second basic cluster in a bipartite NLS ( approximately 3 kcal/mol) as well as the energy of inhibition of the importin alpha importin beta-binding domain ( approximately 3 kcal/mol) were also measured. These data allow the generation of an energetic scale of nuclear localization sequences based on a peptide's affinity for the importin alpha-importin beta complex. On this scale, a functional NLS has a binding constant of approximately 10 nm, whereas a nonfunctional NLS has a 100-fold weaker affinity of 1 microm. Further correlation between the current in vitro data and in vivo function will provide the foundation for a comprehensive quantitative model of protein import.
Highlights
The best characterized mechanism for translocation across the nuclear envelope is protein import which depends on the “classical” nuclear localization signal (NLS)1 [7]
The importin -bound importin ␣ is free to bind to the NLS cargo, and the ternary complex is translocated through the nuclear pore via interactions between importin  and the nucleoporins [15, 16]
The definition of a nuclear localization signal sequence is somewhat vague owing to the diversity of sequences that can apparently act as a functional NLS [7]
Summary
Generation of NLS-GFP Variants and Protein Purification—Various NLS sequences were cloned as in-frame N-terminal fusions to the green fluorescent protein (GFP) through polymerase chain reaction in a pET28a (Novagen) expression vector. The relative free energy contributions reported here were generated using a number of reasonable methods based on approximations In this calculation, we estimate the change in the binding energy, or ⌬⌬G, when a residue of the NLS is substituted with alanine. To obtain a crude estimation of the theoretical contributions of various terms to the relative binding energies of variant NLS sequences, the relative changes in buried surface area, electrostatic interactions, and helicity for each NLS variant was calculated as follows. If a reasonable fit is achieved, the resulting terms Csurf⌬⌬(buried surface), Celec⌬⌬(electrostatics), and Chel⌬⌬(helicity) give the relative contributions of each energy term to the binding of each residue of the NLS to importin ␣
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