Glycosylation is one of the most common posttranslational modifications to occur in protein biosynthesis, yet its effect on the thermodynamics and kinetics of proteins is poorly understood. A minimalist model based on the native protein topology, in which each amino acid and sugar ring was represented by a single bead, was used to study the effect of glycosylation on protein folding. We studied in silico the folding of 63 engineered SH3 domain variants that had been glycosylated with different numbers of conjugated polysaccharide chains at different sites on the protein's surface. Thermal stabilization of the protein by the polysaccharide chains was observed in proportion to the number of attached chains. Consistent with recent experimental data, the degree of thermal stabilization depended on the position of the glycosylation sites, but only very weakly on the size of the glycans. A thermodynamic analysis showed that the origin of the enhanced protein stabilization by glycosylation is destabilization of the unfolded state rather than stabilization of the folded state. The higher free energy of the unfolded state is enthalpic in origin because the bulky polysaccharide chains force the unfolded ensemble to adopt more extended conformations by prohibiting formation of a residual structure. The thermodynamic stabilization induced by glycosylation is coupled with kinetic stabilization. The effects introduced by the glycans on the biophysical properties of proteins are likely to be relevant to other protein polymeric conjugate systems that regularly occur in the cell as posttranslational modifications or for biotechnological purposes.

Intrinsically disordered regions, terminal tails, and flexible linkers are abundant in DNA-binding proteins and play a crucial role by increasing the affinity and specificity of DNA binding. Disordered tails often undergo a disorder-to-order transition during interactions with DNA and improve both the kinetics and thermodynamics of specific DNA binding. The DNA search by proteins that interact nonspecifically with DNA can be supported by disordered tails as well. The disordered tail may increase the overall protein–DNA interface and thus increase the affinity of the protein to the DNA and its sliding propensity while slowing linear diffusion. The exact effect of the disordered tails on the sliding rate depends on the degree of positive charge clustering, as has been shown for homeodomains and p53 transcription factors. The disordered tails, which may be viewed as DNA recognizing subdomains, can facilitate intersegment transfer events that occur via a “monkey bar” mechanism in which the domains bridge two different DNA fragments simultaneously. The “monkey bar” mechanism can be facilitated by internal disordered linkers in multidomain proteins that mediate the cross-talks between the constituent domains and especially their brachiation dynamics and thus their overall capability to search DNA efficiently. The residue sequence of the disordered tails has unique characteristics that were evolutionarily selected to achieve the optimized function that is unique to each protein. Perturbation of the electrostatic characteristics of the disordered tails by post-translational modifications, such as acetylation and phosphorylation, may affect protein affinity to DNA and therefore can serve to regulate DNA recognition. Modifying the disordered protein tails or the flexibility of the inter-domain linkers of multidomain proteins may affect the cross-talk between the constituent domains so as to facilitate the search kinetics of non-specific DNA sequences and increase affinity to the specific sequences.

Rapid recognition of DNA target sites involves facilitated diffusion through which alternative sites are searched on genomic DNA. A key mechanism facilitating the localization of the target by a DNA-binding protein (DBP) is one-dimensional diffusion (sliding) in which electrostatic forces attract the protein to the DNA. As the protein reaches its target DNA site, it switches from purely electrostatic binding to a specific set of interactions with the DNA bases that also involves hydrogen bonding and van der Waals forces. High overlap between the DBP patches used for nonspecific and specific interactions with DNA may enable an immediate transition between the two binding modes following target site localization. By contrast, an imperfect overlap may result in greater frustration between the two potentially competing binding modes and consequently slower switching between them. A structural analysis of 125 DBPs indicates frustration between the two binding modes that results in a large difference between the orientations of the protein to the DNA when it slides compared to when it specifically interacts with DNA. Coarse-grained molecular dynamics simulations of in silico designed peptides comprising the full range of frustrations between the two interfaces show slower transition from nonspecific to specific DNA binding as the overlap between the patches involved in the two binding modes decreases. The complex search kinetics may regulate the search by eliminating trapping of the protein in semispecific sites while sliding.

Intrinsically disordered tails are common in DNA-binding proteins and can affect their search efficiency on nonspecific DNA by promoting the brachiation dynamics of intersegment transfer. During brachiation, the protein jumps between distant DNA regions via an intermediate state in which the tail and globular moieties are bound to different DNA segments. While the disordered tail must be long and positively charged to facilitate DNA search, the effect of its residue sequence on brachiation is unknown. We explored this issue using the NK-2 and Antp homeodomain transcription factors. We designed 566 NK-2 tail-variants and 55 Antp tail-variants having different net charges and positive charge distributions and studied their dynamics and DNA search efficiencies using coarse-grained molecular dynamics simulations. More intersegment transfers occur when the tail is moderately positively charged and the positive charges are clustered together in the middle of the tail or towards its N terminus. The presence of a negatively charged residue does not significantly affect protein brachiation, although it is likely that the presence of many negatively charged residues will complicate the DNA search mechanism. A bioinformatic analysis of 1,384 wild-type homeodomains illustrates that the charge composition and distribution in their N-tail sequences are consistent with an optimal charge pattern to promote intersegment transfer. Our study thus indicates that the residue sequence of the disordered tails of DNA-binding proteins has unique characteristics that were evolutionarily selected to achieve optimized function and suggests that the sequence-structure-function paradigm known for structured proteins is valid for intrinsically disordered proteins as well.

Egr-1 is an inducible transcription factor that recognizes 9-bp target DNA sites via three zinc finger domains and activates genes in response to cellular stimuli such as synaptic signals and vascular stresses. Using spectroscopic and computational approaches, we have studied structural, dynamic, and kinetic aspects of the DNA-scanning process in which Egr-1 is nonspecifically bound to DNA and perpetually changes its location on DNA. Our NMR data indicate that Egr-1 undergoes highly dynamic domain motions when scanning DNA. In particular, the zinc finger 1 (ZF1) of Egr-1 in the nonspecific complex is mainly dissociated from DNA and undergoes collective motions on a nanosecond timescale, whereas zinc fingers 2 and 3 (ZF2 and ZF3, respectively) are bound to DNA. This was totally unexpected because the previous crystallographic studies of the specific complex indicated that all of Egr-1’s three zinc fingers are equally involved in binding to a target DNA site. Mutations that are expected to enhance ZF1’s interactions with DNA and with ZF2 were found to reduce ZF1’s domain motions in the nonspecific complex suggesting that these interactions dictate the dynamic behavior of ZF1. By experiment and computation, we have also investigated kinetics of Egr-1’s translocation between two nonspecific DNA duplexes. Our data on the wild type and mutant proteins suggest that the domain dynamics facilitate Egr-1’s intersegment transfer that involves transient bridging of two DNA sites. These results shed light on asymmetrical roles of the zinc finger domains for Egr-1 to scan DNA efficiently in the nucleus.