For mapping energetic interactions in proteins, a technique was developed that uses evolutionary data for a protein family to measure statistical interactions between amino acid positions. For the PDZ domain family, this analysis predicted a set of energetically coupled positions for a binding site residue that includes unexpected long-range interactions. Mutational studies confirm these predictions, demonstrating that the statistical energy function is a good indicator of thermodynamic coupling in proteins. Sets of interacting residues form connected pathways through the protein fold that may be the basis for efficient energy conduction within proteins.

A fundamental goal in cellular signaling is to understand allosteric communication, the process by which signals originating at one site in a protein propagate reliably to affect distant functional sites. The general principles of protein structure that underlie this process remain unknown. Here, we describe a sequence-based statistical method for quantitatively mapping the global network of amino acid interactions in a protein. Application of this method for three structurally and functionally distinct protein families (G protein-coupled receptors, the chymotrypsin class of serine proteases and hemoglobins) reveals a surprisingly simple architecture for amino acid interactions in each protein family: a small subset of residues forms physically connected networks that link distant functional sites in the tertiary structure. Although small in number, residues comprising the network show excellent correlation with the large body of mechanistic data available for each family. The data suggest that evolutionarily conserved sparse networks of amino acid interactions represent structural motifs for allosteric communication in proteins.

ABC (ATP-binding cassette) proteins constitute a large family of membrane proteins that actively transport a broad range of substrates. Cystic fibrosis transmembrane conductance regulator (CFTR), the protein dysfunctional in cystic fibrosis, is unique among ABC proteins in that its transmembrane domains comprise an ion channel. Opening and closing of the pore have been linked to ATP binding and hydrolysis at CFTR's two nucleotide-binding domains, NBD1 and NBD2 (see, for example, refs 1, 2). Isolated NBDs of prokaryotic ABC proteins dimerize upon binding ATP, and hydrolysis of the ATP causes dimer dissociation. Here, using single-channel recording methods on intact CFTR molecules, we directly follow opening and closing of the channel gates, and relate these occurrences to ATP-mediated events in the NBDs. We find that energetic coupling between two CFTR residues, expected to lie on opposite sides of its predicted NBD1-NBD2 dimer interface, changes in concert with channel gating status. The two monitored side chains are independent of each other in closed channels but become coupled as the channels open. The results directly link ATP-driven tight dimerization of CFTR's cytoplasmic nucleotide-binding domains to opening of the ion channel in the transmembrane domains. This establishes a molecular mechanism, involving dynamic restructuring of the NBD dimer interface, that is probably common to all members of the ABC protein superfamily.

Classical studies show that for many proteins, the information required for specifying the tertiary structure is contained in the amino acid sequence. Here, we attempt to define the sequence rules for specifying a protein fold by computationally creating artificial protein sequences using only statistical information encoded in a multiple sequence alignment and no tertiary structure information. Experimental testing of libraries of artificial WW domain sequences shows that a simple statistical energy function capturing coevolution between amino acid residues is necessary and sufficient to specify sequences that fold into native structures. The artificial proteins show thermodynamic stabilities similar to natural WW domains, and structure determination of one artificial protein shows excellent agreement with the WW fold at atomic resolution. The relative simplicity of the information used for creating sequences suggests a marked reduction to the potential complexity of the protein-folding problem.

Thermodynamic measurements of ion binding to the Streptomyces lividans K+ channel were carried out using isothermal titration calorimetry, whereas atomic structures of ion-bound and ion-free conformations of the channel were characterized by x-ray crystallography. Here we use these assays to show that the ion radius dependence of selectivity stems from the channel's recognition of ion size (i.e., volume) rather than charge density. Ion size recognition is a function of the channel's ability to adopt a very specific conductive structure with larger ions (K+, Rb+, Cs+, and Ba2+) bound and not with smaller ions (Na+, Mg2+, and Ca2+). The formation of the conductive structure involves selectivity filter atoms that are in direct contact with bound ions as well as protein atoms surrounding the selectivity filter up to a distance of 15 Å from the ions. We conclude that ion selectivity in a K+ channel is a property of size-matched ion binding sites created by the protein structure.

Split inteins are parasitic genetic elements frequently found inserted into reading frames of essential proteins. Their association and excision restore host protein function through a protein self-splicing reaction. They have gained an increasingly important role in the chemical modification of proteins to create cyclical, segmentally labeled, and fluorescently tagged proteins. Ideally, inteins would seamlessly splice polypeptides together with no remnant sequences and at high efficiency. Here, we describe experiments that identify the branched intermediate, a transient step in the overall splicing reaction, as a key determinant of the splicing efficiency at different splice-site junctions. To alter intein specificity, we developed a cellbased selection scheme to evolve split inteins that splice with high efficiency at different splice junctions and at higher temperatures. Mutations within these evolved inteins occur at sites distant from the active site. We present a hypothesis that a network of conserved coevolving amino acids in inteins mediates these long-range effects. directed evolution ͉ protein ligation ͉ Crk-II ͉ SCA ͉ coevolution P rotein function is modulated by a variety of posttranslational modifications, such as phosphorylation, ubiquitylation, and methylation (1). One of the most dramatic posttranslational modifications is protein splicing, an autocatalytic process in which an intervening polypeptide sequence, termed an intein, is excised from a precursor protein with concomitant splicing of the franking sequences, known as exteins. Protein splicing has proven to be a highly versatile process for methods development and forms the hub of several protein engineering technologies (2). Inteins have been successfully used in protein semisynthesis to create posttranslationally modified proteins (2, 3), in the cyclization of peptides to create small molecule toxins (4), in plant biotechnology to reconstruct proteins in vivo (5), in the segmental labeling of proteins for NMR studies (6), and in protein semisynthesis in living cells (7).Inteins come in 2 flavors-cis splicing inteins are single polypeptides that are embedded in a host protein, whereas trans-splicing inteins (herein called split inteins) are separate polypeptides that mediate protein splicing after the intein pieces and their protein cargo associate (8, 9) (Fig. 1A). Despite the many applications of split inteins in chemical biology and protein chemistry, they are plagued with various idiosyncratic parameters that limit their more general use. Most importantly, the 2 parts of the naturally split inteins associate and typically splice at a C-terminal junction containing the canonical ''CFN'' tripeptide sequence (10, 11), which are the first 3 amino acids of the C-extein sequence (Fig. 1 A). This tripeptide remains in the product after splicing, meaning that it will most often be a mutant protein. It is currently unclear why this "CFN" sequence is required for efficient protein trans-splicing. In contrast, work from several laboratories, incl...

Members of the G protein superfamily contain nucleotide-dependent switches that dictate the specificity of their interactions with binding partners. Using a sequence-based method termed statistical coupling analysis (SCA), we have attempted to identify the allosteric core of these proteins, the network of amino acid residues that couples the domains responsible for nucleotide binding and protein-protein interactions. One-third of the 38 residues identified by SCA were mutated in the G protein G s␣, and the interactions of guanosine 5-3-O-(thio)triphosphate-and GDPbound mutant proteins were tested with both adenylyl cyclase (preferential binding to GTP-G s␣) and the G protein ␤␥ subunit complex (preferential binding to GDP-G s␣ ). A two-state allosteric model predicts that mutation of residues that control the equilibrium between GDP-and GTP-bound conformations of the protein will cause the ratio of affinities of these species for adenylyl cyclase and G␤␥ to vary in a reciprocal fashion. Observed results were consistent with this prediction. The network of residues identified by the SCA appears to comprise a core allosteric mechanism conferring nucleotide-dependent switching; the specific features of different G protein family members are built on this core.

A crystal structure of a member of the UbiA family of membrane-embedded prenyltransferases reveals the architecture of the active site and suggests a possible mechanism for catalysis.