A variety of methods exist for the design or selection of antibodies and other proteins that recognize the water-soluble regions of proteins; however, companion methods for targeting transmembrane (TM) regions are not available. Here, we describe a method for the computational design of peptides that target TM helices in a sequence-specific manner. To illustrate the method, peptides were designed that specifically recognize the TM helices of two closely related integrins (alphaIIbbeta3 and alphavbeta3) in micelles, bacterial membranes, and mammalian cells. These data show that sequence-specific recognition of helices in TM proteins can be achieved through optimization of the geometric complementarity of the target-host complex.

Single-span transmembrane (TM) helices have structural and functional roles well beyond serving as mere anchors to tether water-soluble domains in the vicinity of the membrane. They frequently direct the assembly of protein complexes and mediate signal transduction in ways analogous to small modular domains in water-soluble proteins. This review highlights different sequence and structural motifs that direct TM assembly and discusses their roles in diverse biological processes. We believe that TM interactions are potential therapeutic targets, as evidenced by natural proteins that modulate other TM interactions and recent developments in the design of TM-targeting peptides.

Nature has evolved several unique biomineralization strategies to direct the synthesis and growth of inorganic materials. These natural systems are complex, involving the interaction of multiple biomolecules to catalyze biomineralization and template growth. Herein we describe the first report to our knowledge of a single enzyme capable of both catalyzing mineralization in otherwise unreactive solution and of templating nanocrystal growth. A recombinant putative cystathionine γ-lyase (smCSE) mineralizes CdS from an aqueous cadmium acetate solution via reactive H 2 S generation from L-cysteine and controls nanocrystal growth within the quantum confined size range. The role of enzymatic nanocrystal templating is demonstrated by substituting reactive Na 2 S as the sulfur source. Whereas bulk CdS is formed in the absence of the enzyme or other capping agents, nanocrystal formation is observed when smCSE is present to control the growth. This dualfunction, single-enzyme, aerobic, and aqueous route to functional material synthesis demonstrates the powerful potential of engineered functional material biomineralization.cadmium sulfide | quantum dot | biomineralization | enzyme | nanoparticle B iological systems have evolved a diverse array of mechanisms to synthesize inorganic materials from aqueous solutions under ambient conditions. This inherent control over material properties has created interest in using these biological routes to synthesize materials (1-3) such as biosilica from sponges and diatoms (4-8), biogenic CaCO 3 from mollusks (9-12), and magnetic particles from magnetotactic bacteria (13-15). Designing a biomineralization strategy requires control of both the material composition and structure; in nature, this control is typically achieved through the assembly of a multiprotein complex, including both structuredirecting proteins and proteins responsible for mineralization of a specific composition. In the current work, we demonstrate the reduction of this complexity to its simplest form: a single enzyme capable of both catalyzing CdS mineralization and controlling particle size within the quantum confined size range to form functional biomineralized CdS quantum dots.Two of the most studied biomineralization proteins are perlucin and silicatein. Perlucin (16) has been shown to mineralize crystalline forms of CaCO 3 , a common structural material that constitutes the shell of many marine organisms, in the form of organic-inorganic composites. The role of the nacre protein perlucin in crystallite templating has been elucidated through experiments demonstrating crystallite formation in the presence of purified perlucin, and perlucin selectively being removed from solution during crystallite formation in the presence of a mixture of water-soluble, nacre-associated proteins (17). Native silicatein harvested from sea sponge or engineered forms produced recombinantly are active for biomineralization of silica and titania into structures that are amorphous or crystalline (7,18,19). In particular, biomineralizatio...

Summary. Integrins are a ubiquitous family of non-covalently associated a/b transmembrane heterodimers linking extracellular ligands to intracellular signaling pathways [1] [Cell, 2002; 110: 673]. Platelets contain five integrins, three b1 integrins that mediate platelet adhesion to the matrix proteins collagen, fibronectin and laminin, and the b3 integrins avb3 and aIIbb3[2] [J Clin Invest, 2005; 115: 3363]. While there are only several hundred avb3 molecules per platelet, avb3 mediates platelet adhesion to osteopontin and vitronectin in vitro [3] [J Biol Chem, 1997; 272: 8137]; whether this occurs in vivo remains unknown. By contrast, the 80 000 aIIbb3 molecules on agoniststimulated platelets bind fibrinogen, von Willebrand factor, and fibronectin, mediating platelet aggregation when the bound proteins crosslink adjacent platelets [2] [J Clin Invest, 2005; 115: 3363]. Although platelet integrins are poised to shift from resting to active conformations, tight regulation of their activity is essential to prevent the formation of intravascular thrombi. This review focuses on the structure and function of the intensively studied b3 integrins, in particular aIIbb3, but reference will be made to other integrins where relevant.Keywords: glycoprotein IIb/IIIa, integrins, protein structure. The extracellular domain of b3 integrinsElectron microscopy (EM) of aIIbb3 isolated from platelets revealed an 8 · 12 nm nodular headpiece containing its ligand binding site and two 18 nm flexible legs containing transmembrane (TM) and cytoplasmic domains extending from one side [4]. Further, crystal structures for the extracellular portions of avb3 and aIIbb3 have revealed a complex domain structure that rearranges as the integrins switch from inactive resting to active ligand-binding conformations (Fig. 1) [5][6][7][8].The extracellular portions of aIIb and av are similar, consisting of an amino-terminal b-propeller domain followed by a ÔthighÕ and two ÔcalfÕ domains, whereas b3 is substantially more complicated [5,7]. Its amino-terminal portion consists of two tandem nested domains: a bA domain whose fold resembles that of integrin a subunit I-domains, which is inserted into a ÔhybridÕ domain whose fold is similar to an I-set Ig domain; the hybrid domain in turn is inserted into a PSI (plexin, semaphorin, integrin) domain that contains the N-terminus of b3 [7,9]. The C-terminus of the PSI domain is continuous with four tandem EGF-like repeats that make up the ÔlegÕ of b3, as well as a unique carboxyl-terminal bTD domain [8]. av and aIIb interact non-covalently with b3 via an interface between the a subunit b-propeller and the b3 bA domains that resembles the interface between the Ga and Gb subunits of G proteins and forms the surface for ligand binding [5,7].A surprising finding of the crystal structure of the extracellular portion of avb3 was a severe bend at ÔkneesÕ or ÔgenusÕ located between the first and second EGF-like repeats of b3 and the thigh and first calf domains of av [5,10]. Recent high resolution crystal structu...

Important progress has been made in recent years toward developing a molecular-level understanding of protein phase behavior in terms of the osmotic second virial coefficient, a thermodynamic parameter that characterizes pairwise protein interactions. Yet there has been little practical application of this knowledge to the field of protein crystallization, largely because of the difficult and time-consuming nature of traditional techniques for characterizing protein interactions. Self-interaction chromatography has recently been proposed as a highly efficient method for measuring the osmotic second virial coefficient. The utility of the technique is examined in this work by characterizing virial coefficients for ribonuclease A under 59 solution conditions using several crystallization additives, including PEG, sodium chloride, ammonium sulfate, and propanol. The virial coefficient measurements show some counterintuitive trends and shed light on the previous difficulties in crystallizing ribonuclease A. Crystallization experiments at the corresponding solution conditions were conducted by using ultracentrifugal crystallization. Using this methodology, ribonuclease A crystals were obtained under conditions for which the virial coefficients fell within the "crystallization slot." Crystallographic characterization showed that the crystals diffract to high resolution. Metastable crystals were also obtained for conditions outside, but near, the "crystallization slot," and they could also be frozen and used to collect structural information.