Bryan W. Berger

Computational Design of Peptides That Target Transmembrane Helices

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 (αIIbβ3 and αvβ3) 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.

Protein-Protein Interactions in the Membrane: Sequence, Structural, and Biological Motifs

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.

The structure and function of platelet integrins

Summary.u2002 Integrins are a ubiquitous family of non‐covalently associated α/β transmembrane heterodimers linking extracellular ligands to intracellular signaling pathways [ 1 ] [Cell, 2002; 110: 673]. Platelets contain five integrins, three β1 integrins that mediate platelet adhesion to the matrix proteins collagen, fibronectin and laminin, and the β3 integrins αvβ3 and αIIbβ3 [ 2 ] [J Clin Invest, 2005; 115: 3363]. While there are only several hundred αvβ3 molecules per platelet, αvβ3 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 80u2003000 αIIbβ3 molecules on agonist‐stimulated 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 β3 integrins, in particular αIIbβ3, but reference will be made to other integrins where relevant.

Self-interaction chromatography: a novel screening method for rational protein crystallization

The osmotic second virial coefficient, B(22), has become the quantity most widely used in developing a rational understanding of protein crystallization. In this work a novel method of measuring B22 using self-interaction chromatography (SIC) is presented that is at least an order of magnitude more efficient than traditional characterization methods, such as static light scattering. It is shown that SIC measurements of second virial coefficients for BSA are in quantitative agreement with static light scattering results. The measured virial coefficient for both BSA and myoglobin reveal a surprisingly narrow range of concentrations of ammonium sulfate that promote weakly attractive interactions that are optimal for crystallization. Using the virial coefficient information, myoglobin crystals were obtained by ultracentrifugal crystallization in a rational and rapid manner.

Predictive crystallization of ribonuclease A via rapid screening of osmotic second virial coefficients

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. Proteins 2003;50:303–311.

Effects of additives on surfactant phase behavior relevant to bacteriorhodopsin crystallization

The interactions leading to crystallization of the integral membrane protein bacteriorhodopsin solubilized in n‐octyl‐β‐D‐glucoside were investigated. Osmotic second virial coefficients (B22) were measured by self‐interaction chromatography using a wide range of additives and precipitants, including polyethylene glycol (PEG) and heptane‐1,2,3‐triol (HT). In all cases, attractive protein–detergent complex (PDC) interactions were observed near the surfactant cloud point temperature, and there is a correlation between the surfactant cloud point temperatures and PDC B22 values. Light scattering, isothermal titration calorimetry, and tensiometry reveal that although the underlying reasons for the patterns of interaction may be different for various combinations of precipitants and additives, surfactant phase behavior plays an important role in promoting crystallization. In most cases, solution conditions that led to crystallization fell within a similar range of slightly negative B22 values, suggesting that weakly attractive interactions are important as they are for soluble proteins. However, the sensitivity of the cloud point temperatures and resultant coexistence curves varied significantly as a function of precipitant type, which suggests that different types of forces are involved in driving phase separation depending on the precipitant used.

The role of protein and surfactant interactions in membrane-protein crystallization.

The interactions leading to crystallization of the integral membrane protein bacteriorhodopsin solubilized in n-octyl-beta-D-glucoside were investigated. Osmotic second virial coefficients were measured by self-interaction chromatography in the presence of sodium malonate, sodium formate and ammonium sulfate. Attractive protein-detergent complex (PDC) interactions were observed as the surfactant cloud-point temperature was approached for each salt, suggesting that surfactant interactions may play an important role in promoting PDC crystallization. Dynamic light scattering and tensiometry measurements show that the interaction trends are strongly influenced by micelle structure and surfactant phase behavior, both of which are sensitive to salt and surfactant concentration. Overall, detailed investigations using a combination of experimental techniques can provide insight into the complex nature of PDC interactions, which is essential to developing rational approaches to membrane-protein crystallization.