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The ability to design proteins with high affinity and selectivity for any given small molecule is a rigorous test of our understanding of the physiochemical principles that govern molecular recognition. Attempts to rationally design ligand-binding proteins have met with little success, however, and the computational design of protein-small-molecule interfaces remains an unsolved problem (1). Current approaches for designing ligand-binding proteins for medical (2) and biotechnological uses rely on raising antibodies against a target antigen in immunized animals (3,4) and/or performing laboratory-directed evolution of proteins with an existing low affinity for the desired ligand (5-7), neither of which allows complete control over the interactions involved in binding. Here we describe a general computational method for designing pre-organized and shape complementary small-molecule-binding sites, and use it to generate protein binders to the steroid digoxigenin (DIG). Of seventeen experimentally characterized designs, two bind DIG; the model of the higher affinity binder has the most energetically favourable and pre-organized interface in the design set. A comprehensive binding-fitness landscape of this design, generated by library selections and deep sequencing, was used to optimize its binding affinity to a picomolar level, and X-ray co-crystal structures of two variants show atomic-level agreement with the corresponding computational models. The optimized binder is selective for DIG over the related steroids digitoxigenin, progesterone and β-oestradiol, and this steroid binding preference can be reprogrammed by manipulation of explicitly designed hydrogen-bonding interactions. The computational design method presented here should enable the development of a new generation of biosensors, therapeutics and diagnostics., Computational design could provide a general approach for creating new small molecule binding proteins with rationally programmed specificities. Structural and biophysical characterization of previous computationally designed ligand-binding proteins revealed numerous [...]

How the interplay between protein structure and internal dynamics regulates protein function is poorly understood. Often, ligand binding, post-translational modifications and mutations modify protein activity in a manner that is [...]

Allosteric regulation is used as a very efficient mechanism to control protein activity in most biological processes, including signal transduction, metabolism, catalysis and gene regulation (1-6). Allosteric proteins can exist in several conformational states with distinct binding or enzymatic activity. Effectors are considered to function in a purely structural manner by selectively stabilizing a specific conformational state, thereby regulating protein activity. Here we show that allosteric proteins can be regulated predominantly by changes in their structural dynamics. We have used NMR spectroscopy and isothermal titration calorimetry to characterize cyclic AMP (cAMP) binding to the catabolite activator protein (CAP), a transcriptional activator that has been a prototype for understanding effector-mediated allosteric control of protein activity (7). cAMP switches CAP from the 'off' state (inactive), which binds DNA weakly and nonspecifically, to the 'on' state (active), which binds DNA strongly and specifically. In contrast, cAMP binding to a single CAP mutant, CAP-S62F, fails to elicit the active conformation; yet, cAMP binding to CAP-S62F strongly activates the protein for DNA binding. NMR and thermodynamic analyses show that despite the fact that CAP-S62F-[cAMP.sub.2] adopts the inactive conformation, its strong binding to DNA is driven by a large conformational entropy originating in enhanced protein motions induced by DNA binding. The results provide strong evidence that changes in protein motions may activate allosteric proteins that are otherwise structurally inactive., The cAMP-mediated activation of CAP for DNA binding (8-10) is a characteristic example of the widely held view that allosteric regulation is predominantly structural in origin (6,11). The DNA-binding domain [...]

Extra-cytoplasmic polypeptides are usually synthesized as 'preproteins' carrying amino-terminal, cleavable signal peptides (1) and secreted across membranes by translocases. The main bacterial translocase comprises the SecYEG protein-conducting channel and the peripheral ATPase motor SecA (2,3). Most proteins destined for the periplasm and beyond are exported post-translationally by SecA (2,3). Preprotein targeting to SecA is thought to involve signal peptides (4) and chaperones like SecB (5,6). Here we show that signal peptides have a new role beyond targeting: they are essential allosteric activators of the translocase. On docking on their binding groove on SecA, signal peptides act in trans to drive three successive states: first, 'triggering' that drives the translocase to a lower activation energy state; second, 'trapping'that engages non-native preprotein mature domains docked with high affinity on the secretion apparatus; and third, 'secretion' during which trapped mature domains undergo several turnovers of translocation in segments (7). A significant contribution by mature domains renders signal peptides less critical in bacterial secretory protein targeting than currently assumed. Rather, it is their function as allosteric activators of the translocase that renders signal peptides essential for protein secretion. A role for signal peptides and targeting sequences as allosteric activators may be universal in protein translocases., We sought to dissect the individual contributions of signal peptides and mature domains to membrane targeting and to post-targeting translocation steps. Because SecB is not universal or essential (6,8), we [...]

The Lactor repressor system is used to recognize deoxyribonucleic acid from nuclear magnetic resonance studies. The most challenging issue for specific protien-DNA recognition is that the target sequence is immersed in a huge molar excess of nonspecific DNA sequences.

Research has been conducted on the carbon and oxygen shieldings and oxygen isotope effects on carbon shieldings of carbon monoxide in solvents. Comparison with hemoproteins is presented.

57Fe NMR spectroscopy is used in measuring porphyrin ruffling in hexacoordinated carbonmonoxy heme model compounds in solution. Ruffling occurs when opposite pyrrole rings are counterrotated so that the meso carbon atoms of each pyrrole ring are alternately displaced above and below the mean porphyrin plane. Results indicated good correlation between delta(57Fe) and the absolute crystallographic average displacement of the meso carbon atoms relative to the porphyrin core mean plane. It was also noted that 57Fe shieldings are excellent protocols in deriving precise structures of heme rings in heme model compounds.

Molecular chaperones act on non-native proteins in the cell to prevent their aggregation, premature folding or misfolding. Different chaperones often exert distinct effects, such as acceleration or delay of folding, on client proteins via mechanisms that are poorly understood. Here we report the solution structure of SecB, a chaperone that exhibits strong antifolding activity, in complex with alkaline phosphatase and maltose-binding protein captured in their unfolded states. SecB uses long hydrophobic grooves that run around its disk-like shape to recognize and bind to multiple hydrophobic segments across the length of non-native proteins. The multivalent binding mode results in proteins wrapping around SecB. This unique complex architecture alters the kinetics of protein binding to SecB and confers strong antifolding activity on the chaperone. The data show how the different architectures of chaperones result in distinct binding modes with non-native proteins that ultimately define the activity of the chaperone., Author(s): Chengdong Huang [1]; Paolo Rossi [1]; Tomohide Saio [1]; Charalampos G. Kalodimos (corresponding author) [1] Molecular chaperones rescue non-native proteins in the cell from aggregation and assist with their [...]