Amyloid formation plays a role in a wide range of human diseases. The rate and extent of amyloid formation depends on solution conditions including pH and ionic strength. Amyloid fibrils often adopt structures with parallel, in-register β-sheets, which generate quasi-infinite arrays of aligned side chains. These arrangements can lead to significant electrostatic interactions between adjacent polypeptide chains. The effect of ionic strength and ion composition on the kinetics of amyloid formation by islet amyloid polypeptide (IAPP) is examined. IAPP is a basic 37-residue polypeptide responsible for islet amyloid formation in type 2 diabetes. Poisson–Boltzmann calculations revealed significant electrostatic repulsion in a model of the IAPP fibrillar state. The kinetics of IAPP amyloid formation are strongly dependent on ionic strength, varying by more than a factor of 10 over the range of 20 to 600 mM NaCl at pH 8.0, but the effect is not entirely due to Debye screening. At low ionic strength the rate depends strongly on the identity of the anion, nearly varying by a factor of four and scales with the electroselectivity series, implicating anion binding. At high ionic strength the rate varies by only 8% and scales with the Hofmeister series. At intermediate ionic strength no clear trend is detected, likely because of convolution of different effects. The effects of salts on the growth phase and lag phase of IAPP amyloid formation are strongly correlated. At pH 5.5, where the net charge on IAPP is larger, the effect of different anions scales with the electroselectivity series at all salt concentrations.

The use of noncoded amino acids as spectroscopic probes of protein folding and function is growing rapidly, in large part because of advances in the methodology for their incorporation. Recently p-cyanophenylalanine has been employed as a fluorescence and IR probe, as well as a FRET probe to study protein folding, protein-membrane interactions, protein-protein interactions and amyloid formation. The probe has been shown to be exquisitely sensitive to hydrogen bonding interactions involving the cyano group, and its fluorescence quantum yield increases dramatically when it is hydrogen bonded. However, a detailed understanding of the factors which influence its fluorescence is required to be able to use this popular probe accurately. Here we demonstrate the recombinant incorporation of p-cyanophenylalanine in the N-terminal domain of the ribosomal protein L9. Native state fluorescence is very low, which suggests that the group is sequestered from solvent; however, IR measurements and molecular dynamics simulations show that the cyano group is exposed to solvent and forms hydrogen bonds to water. Analysis of mutant proteins and model peptides demonstrates that the reduced native state fluorescence is caused by the effective quenching of p-cyanophenylalanine fluorescence via FRET to tyrosine side-chains. The implications for the interpretation of p-cyanophenylalanine fluorescence measurements and FRET studies are discussed.

The rational modification of protein stability is an important goal of protein design. Protein surface electrostatic interactions are not evolutionarily optimized for stability and are an attractive target for the rational redesign of proteins. We show that surface charge mutants can exert stabilizing effects in distinct and unanticipated ways, including ones that are not predicted by existing methods, even when only solvent-exposed sites are targeted. Individual mutation of three solvent-exposed lysines in the villin headpiece subdomain significantly stabilizes the protein, but the mechanism of stabilization is very different in each case. One mutation destabilizes native-state electrostatic interactions but has a larger destabilizing effect on the denatured state, a second removes the desolvation penalty paid by the charged residue, whereas the third introduces unanticipated native-state interactions but does not alter electrostatics. Our results show that even seemingly intuitive mutations can exert their effects through unforeseen and complex interactions.atomistic simulations | pH-dependent stability | protein pKa measurements | nuclear magnetic resonance | protein biophysics T he mutation of charged surface residues to enhance electrostatics is a popular approach in protein engineering (1). Target residues are often selected using estimates of the protein electrostatic potential (2). This approach relies on identifying residues that are involved in unfavorable electrostatic interactions, typically with residues of the same charge, or on identifying sites where new favorable electrostatic interactions can be introduced (3). Solvent-exposed charged residues are thought to not be involved in critical packing interactions in the native state, to not suffer large desolvation penalties upon protein folding, nor to make significant interactions in the denatured-state ensemble (DSE). Thus, residues to be targeted are typically chosen on the basis of calculations of protein native-state ensemble (NSE) electrostatics. Methods ranging from simple inspection of the protein surface, modified Tanford-Kirkwood approaches (3, 4) and Poisson-Boltzmann (PB) calculations have been used (5-7). Irrespective of the details, the general strategy is based on the assumption that surface electrostatic interactions can be modified without altering other native-state interactions and without altering DSE energetics. An attractive feature of these approaches is that they are computationally inexpensive and, unlike selectionbased methods or directed evolution, involve the generation of a limited number of mutants. Any increase in stability is generally assumed to result from modification of NSE electrostatics. We show that this approach leads to complicated and unanticipated results, even for very simple proteins.We use the villin headpiece subdomain HP36 as our model system. HP36 is a small three-helix protein that has become an extremely popular model system for experimental and computational studies of protein folding, owing to its ...

Cyanovirin-N is a cyanobacterial lectin with potent antiviral activity, and has been the focus of extensive pre-clinical investigation as a potential prophylactic for the prevention of the sexual transmission of the human immunodeficiency virus (HIV). Here we present a detailed analysis of carbohydrate recognition by this important protein, using a combination of computational methods, including extensive molecular dynamics simulations and Molecular-Mechanics/ Poisson–Boltzmann/Surface-Area (MM/PBSA) energetic analysis. The simulation results strongly suggest that the observed tendency of wildtype CVN to form domain-swapped dimers is the result of a previously unidentified cis-peptide bond present in the monomeric state. The energetic analysis additionally indicates that the highest-affinity ligand for CVN characterized to date (α-Man-(1,2)-α-Man-(1,2)-α-Man) is recognized asymmetrically by the two binding sites. Finally, we are able to provide a detailed map of the role of all binding site functional groups (both backbone and side chain) to various aspects of molecular recognition: general affinity for cognate ligands, specificity for distinct oligosaccharide targets and the asymmetric recognition of α-Man-(1,2)-α-Man-(1,2)-α-Man. Taken as a whole, these results complement past experimental characterization (both structural and thermodynamic) to provide the most complete understanding of carbohydrate recognition by CVN to date. The results also provide strong support for the application of similar approaches to the understanding of other protein–carbohydrate complexes.

Calcium-saturated calmodulin (CaM) binds and influences the activity of a varied collection of target proteins in most cells. This promiscuity underlies CaM's role as a shared participant in calciumdependent signal transduction pathways, but imposes a handicap on popular CaM-based calcium biosensors, which display an undesired tendency to cross-react with cellular proteins. Designed CaM/ target pairs that retain high affinity for one another, but lack affinity for wild-type CaM and its natural interaction partners, would therefore be useful as sensor components, and possibly also as elements of "synthetic" cellular signaling networks. Here we have adopted a rational approach to creating suitably modified CaM/target complexes by using computational design methods to guide parallel site-directed mutagenesis of both binding partners. A hierarchical design procedure was applied to suggest a small number of complementary mutations on CaM and on a peptide ligand derived from skeletal muscle light chain kinase (M13). Experimental analysis showed that the procedure was successful in identifying CaM and M13 mutants with novel specificity for one another. Importantly, the designed complexes retained affinity comparable to the wild-type CaM/M13 complex. These results represent a step toward the creation of CaM and M13 derivatives with specificity fully orthogonal to the wild-type proteins, and show that qualitatively accurate predictions may be obtained from computational methods applied simultaneously to two proteins involved in multiple linked binding equilibria. Figure 1A). (2,3) Calcium-loaded CaM binds to isolated target sequences with affinity comparable to its complexes with the intact proteins, and with dissociation constants often in the nanomolar range. With few exceptions, CaM ligands tend to be highly basic, presenting lysine and arginine residues that form salt-bridge networks with negatively-charged and polar amino-acid side chains bordering the binding cleft on CaM ( Figure 1B). In addition, many CaM targets appear to be anchored by a key pair of bulky groups spaced apart by 2.5 or 3.5 helical turns. (4) Deletion or mutation of these anchor residues dramatically reduces CaMbinding affinity. (5,6) Considerable interest in CaM/peptide interactions has revolved around potential applications in biotechnology. CaM-affinity chromatography (7) The ability to understand and control determinants of binding specificity at the CaM/target interface is important both for biotechnological applications and for appreciation of how CaM transduces metabolic signals via multiple protein interaction networks. Many studies have probed the contributions of both the charged and hydrophobic anchor residues to CaM binding affinity, (5,6,(19)(20)(21)(22)) but relatively few have sought to manipulate specificity through purposeful mutagenesis of one or both binding partners. A step in this direction was taken by Mayo and colleagues,(23,24) and involved the integration of computational and experimental methods to bias CaM spe...

Continuum electrostatic methods are a powerful tool for the analysis and design of biomolecular complexes, with methodologies that allow for the detailed analysis of the electrostatic contributions to binding affinities and procedures for computing the properties of electrostatically optimal ligands. We have applied these methods to the design of improved inhibitors of HIV-1 cell entry. HIV infection of a cell requires viral-cell membrane fusion, an event partially driven by a large-scale conformational change in the viral membrane glycoprotein gp41. This transformation involves the docking of a helix from the C-terminal region of three gp41 chains against a pre-formed trimeric-coiled coil; several protein constructs that inhibit membrane fusion act by binding to an isolated C-terminal helix and blocking the formation of the fusogenic structure. A detailed analysis of the electrostatic contributions to the binding of one such inhibitor (5-Helix) to a C-terminal helix was performed using the X-ray crystal structure of the core of the HIV-1 gp41 ectodomain as a structural model, and several residues on 5-Helix that make substantial contributions to binding, both favorable and unfavorable, were identified. An electrostatic affinity optimization methodology was applied to the side chains of 5-Helix, with the results showing that significant improvements in binding affinity are possible if the electrostatic contributions to the binding free energy are optimized. Several mutations accessible by experimental methods are suggested, with calculated improvements in binding affinity of as much as 500-fold and greater.

Cyanovirin-N (CVN) is an 11-kDa pseudo-symmetric cyanobacterial lectin that has been shown to inhibit infection by the Human Immunodeficiency Virus (HIV) by binding to high-mannose oligosaccharides on the surface of the viral envelope glycoprotein gp120. In this work we describe rationally-designed CVN variants that stabilize the protein fold while maintaining high affinity and selectivity for their glycan targets. Poisson–Boltzmann calculations and protein repacking algorithms were used to select stabilizing mutations in the protein core. By substituting the buried polar side chains of Ser11, Ser20, and Thr61 with aliphatic groups, we stabilized CVN by nearly 12 °C against thermal denaturation, and by 1 m of GuaHCl against chemical denaturation, relative to a previously-characterized stabilized mutant. Glycan microarray binding experiments confirmed that the specificity profile of carbohydrate binding is unperturbed by the mutations, and is identical for all variants. In particular, the variants selectively bound glycans containing the Manα(1→2)Man linkage, which is the known minimal binding unit of CVN. We also report the slow denaturation kinetics of CVN and show that they can complicate thermodynamic analysis; in particular, the unfolding of CVN cannot be described as a fixed two-state transition. Accurate thermodynamic parameters are needed to describe the complicated free energy landscape of CVN, and we provide updated values for CVN unfolding.

The identification of protein mutations that enhance binding affinity may be achieved by computational or experimental means, or by a combination of the two. Sources of affinity enhancement may include improvements to the net balance of binding interactions of residues forming intermolecular contacts at the binding interface, such as packing and hydrogen-bonding interactions. Here we identify noncontacting residues that make substantial contributions to binding affinity and that also provide opportunities for mutations that increase binding affinity of the TEM1 ␤-lactamase (TEM1) to the ␤-lactamase inhibitor protein (BLIP). A region of BLIP not on the direct TEM1-binding surface was identified for which changes in net charge result in particularly large increases in computed binding affinity. Some mutations to the region have previously been characterized, and our results are in good correspondence with this results of that study. In addition, we propose novel mutations to BLIP that were computed to improve binding significantly without contacting TEM1 directly. This class of noncontacting electrostatic interactions could have general utility in the design and tuning of binding interactions.Keywords: continuum electrostatics; electrostatic complementarity; protein binding; protein-protein interactions; protein designThe field of protein design has made substantial advances over the last 20 years, based largely on phrasing the appropriate inverse problem and developing methods capable of addressing inverse design (Drexler 1981;Pabo 1983). Much current protein design work involves the construction of stabilizing protein side-chain arrangements by methods such as dead-end elimination (Desmet et al. 1992;Goldstein 1994;Lasters et al. 1995;Gordon andMayo 1998, 1999;Leach and Lemon 1998;Mendes et al. 1999;De Maeyer et al. 2000;Looger and Hellinga 2001), self-consistent meanfield theory (Koehl and Delarue 1994; Koehl and Levitt 1999a,b;Kono and Saven 2001), simulated annealing (Lee and Subbiah 1991;Hellinga and Richards 1994;Shenkin et al. 1996;Jiang et al. 1997Jiang et al. , 2000, genetic algorithms (Tufféry et al. 1991(Tufféry et al. , 1993(Tufféry et al. , 1997Jones 1994;Desjarlais and Handel 1995), and combinatorial search (Tufféry et al. 1991(Tufféry et al. , 1993(Tufféry et al. , 1997. That is, successful design has been achieved by consideration of detailed atomic interactions and their effect on packing geometry and energetics (Dahiyat and Mayo 1997;Harbury et al. 1998;Calhoun et al. 2003;Kuhlman et al. 2003). The design of protein binding interfaces may be achieved by a similar overall approach, although the additional requirement to treat solvation and electrostatic interactions adds a further layer of complexity (Lee and Tidor 2001a).An alternative strategy that does not demand the same detailed packing of side chains into an exquisite three-dimensional jigsaw puzzle may be desirable in many cases. One such method involves the enhancement of affinity through relatively long-range electrostatic ef...