Vikas Nanda - Academia.edu (original) (raw)

Papers by Vikas Nanda

Methods in enzymology, 2011

The ability to engineer novel proteins using the principles of molecular structure and energetics... more The ability to engineer novel proteins using the principles of molecular structure and energetics is a stringent test of our basic understanding of how proteins fold and maintain structure. The design of protein self-assembly has the potential to impact many fields of biology from molecular recognition to cell signaling to biomaterials. Most progress in computational design of protein self-assembly has focused on α-helical systems, exploring ways to concurrently optimize the stability and specificity of a target state. Applying these methods to collagen self-assembly is very challenging, due to fundamental differences in folding and structure of α- versus triple-helices. Here, we explore various computational methods for designing stable and specific oligomeric systems, with a focus on α-helix and collagen self-assembly.

Proteins: Structure, Function, and Bioinformatics, 2004

Cylcodextrin sugars are cyclic sugars that have a hydrophilic exterior and a hydrophobic center. ... more Cylcodextrin sugars are cyclic sugars that have a hydrophilic exterior and a hydrophobic center. This enables cyclodextrins to solubilize hydrophobic molecules in aqueous media. Cyclodextrins may inhibit aggregation by intercalating surface aromatic residues and competing with interprotein aromatic clusters (pi-pi interactions). In order to investigate this concept, the interaction of hydroxypropyl-beta-cyclodextrin (HPBCD) with melittin is studied with steady-state and time-resolved fluorescence, fluorescence polarization, circular dichroism, and IR spectroscopy. HPBCD inhibits the aggregation of melittin. This inhibition and the spectroscopic results are consistent with the lone aromatic tryptophan of the peptide being intercalated within HPBCD.

Protein science : a publication of the Protein Society, 2012

Outer membrane β-barrel proteins differ from α-helical inner membrane proteins in lipid environme... more Outer membrane β-barrel proteins differ from α-helical inner membrane proteins in lipid environment, secondary structure, and the proposed processes of folding and insertion. It is reasonable to expect that outer membrane proteins may contain primary sequence information specific for their folding and insertion behavior. In previous work, a depth-dependent insertion potential, E(z) , was derived for α-helical inner membrane proteins. We have generated an equivalent potential for TM β-barrel proteins. The similarities and differences between these two potentials provide insight into unique aspects of the folding and insertion of β-barrel membrane proteins. This potential can predict orientation within the membrane and identify functional residues involved in intermolecular interactions.

Methods in molecular biology (Clifton, N.J.), 2013

Protein-protein interactions (PPI) play central roles in biological processes, motivating us to u... more Protein-protein interactions (PPI) play central roles in biological processes, motivating us to understand the structural basis underlying affinity and specificity. In this chapter, we focus on biochemical and computational design strategies of assessing and detecting PPIs of β-barrel outer membrane proteins (OMPs). A few case studies are presented highlighting biochemical techniques used to dissect the energetics of oligomerization and determine amino acids forming the key interactions of the PPI sites. Current computational strategies for detecting/predicting PPIs are introduced, and examples of computational and rational engineering strategies applied to OMPs are presented.

Proteins: Structure, Function, and Bioinformatics, 2005

Characterizing the interactions between amino acid residues and lipid molecules is important for ... more Characterizing the interactions between amino acid residues and lipid molecules is important for understanding the assembly of transmembrane helices and for studying membrane protein folding. In this study we develop TMLIP (Trans-Membrane helix-LIPid), an empirically derived propensity of individual residue types to face lipid membrane based on statistical analysis of highresolution structures of membrane proteins. Lipid accessibilities of amino acid residues within the transmembrane (TM) region of 29 structures of helical membrane proteins are studied with a spherical probe of radius of 1.9 Å. Our results show that there are characteristic preferences for residues to face the headgroup region and the hydrocarbon core region of lipid membrane. Amino acid residues Lys, Arg, Trp, Phe, and Leu are often found exposed at the headgroup regions of the membrane, where they have high propensity to face phospholipid headgroups and glycerol backbones. In the hydrocarbon core region, the strongest preference for interacting with lipids is observed for Ile, Leu, Phe and Val. Small and polar amino acid residues are usually buried inside helical bundles and are strongly lipophobic. There is a strong correlation between various hydrophobicity scales and the propensity of a given residue to face the lipids in the hydrocarbon region of the bilayer. Our data suggest a possibly significant contribution of the lipophobic effect to the folding of membrane proteins. This study shows that membrane proteins have exceedingly apolar exteriors rather than highly polar interiors. Prediction of lipid-facing surfaces of boundary helices using TMLIP1 results in a 54% accuracy, which is significantly better than random (25% accuracy). We also compare performance of TMLIP with another lipid propensity scale, kPROT, and with several hydrophobicity scales using hydrophobic moment analysis. Proteins 2005;59:496 -509.

Proteins: Structure, Function, and Bioinformatics, 2011

The cyclic tetrapyrroles, viz. chlorophylls (Chl), their bacterial analogs bacteriochlorophylls, ... more The cyclic tetrapyrroles, viz. chlorophylls (Chl), their bacterial analogs bacteriochlorophylls, and hemes are ubiquitous cofactors of biological catalysis that are involved in a multitude of reactions. One systematic approach for understanding how Nature achieves functional diversity with only this handful of cofactors is by designing de novo simple and robust protein scaffolds with heme and/or (bacterio)chlorophyll [(B)Chls]-binding sites. This strategy is currently mostly implemented for heme-binding proteins. To gain more insight into the factors that determine heme-/(B)Chl-binding selectivity, we explored the geometric parameters of (B)Chl-binding sites in a nonredundant subset of natural (B)Chl protein structures. Comparing our analysis to the study of a nonredundant database of heme-binding helical histidines by Negron et al. (Proteins 2009;74:400-416), we found a preference for the m-rotamer in (B)Chl-binding helical histidines, in contrast to the preferred t-rotamer in heme-binding helical histidines. This may be used for the design of specific heme-or (B)Chl-binding sites in water-soluble helical bundles, because the rotamer type defines the positioning of the bound cofactor with respect to the helix interface and thus the protein-binding site. Consensus sequences for (B)Chl binding were identified by combining a computational and databasederived approach and shown to be significantly different from the consensus sequences recommended by Negron et al. (Proteins 2009;74:400-416) for heme-binding helical proteins. The insights gained in this work on helix-(B)Chls-binding pockets provide useful guidelines for the construction of reasonable (B)Chl-binding protein templates that can be optimized by computational tools. Proteins 2011; 79:463-476. V V C 2010 Wiley-Liss, Inc. Key words: protein de novo design; tetrapyrrole; histidine rotamers; ligand-binding site; consensus motif. V V C 2010 WILEY-LISS, INC. PROTEINS 463 P. Braun et al.

Proteins: Structure, Function, and Bioinformatics, 2005

In the absence of experimental structural determination, numerous methods are available to indire... more In the absence of experimental structural determination, numerous methods are available to indirectly predict or probe the structure of a target molecule. Genetic modification of a protein sequence is a powerful tool for identifying key residues involved in binding reactions or protein stability. Mutagenesis data is usually incorporated into the modeling process either through manual inspection of model compatibility with empirical data, or through the generation of geometric constraints linking sensitive residues to a binding interface. We present an approach derived from statistical studies of lattice models for introducing mutation information directly into the fitness score. The approach takes into account the phenotype of mutation (neutral or disruptive) and calculates the energy for a given structure over an ensemble of sequences. The structure prediction procedure searches for the optimal conformation where neutral sequences either have no impact or improve stability and disruptive sequences reduce stability relative to wild type. We examine three types of sequence ensembles: information from saturation mutagenesis, scanning mutagenesis, and homologous proteins. Incorporating multiple sequences into a statistical ensemble serves to energetically separate the native state and misfolded structures. As a result, the prediction of structure with a poor force field is sufficiently enhanced by mutational information to improve accuracy. Furthermore, by separating misfolded conformations from the target score, the ensemble energy serves to speed up conformational search algorithms such as Monte Carlo-based methods.

Protein Science, 2005

A peptide designed to form a homo-oligomeric transmembrane helical bundle was reconstituted into ... more A peptide designed to form a homo-oligomeric transmembrane helical bundle was reconstituted into lipid bilayers and studied by using 2 H NMR (nuclear magnetic resonance) with magic angle spinning to confirm that the helical interface corresponds to the interface intended in the design. The peptide belongs to a family of model peptides derived from a membrane-solubilized version of the water-soluble coiled-coil GCN4-P1. The variant studied here contains two asparagines thought to engage in interhelical hydrogen bonding critical to the formation of a stable trimer. For the NMR studies, three different peptides were synthesized, each with one of three consecutive leucines in the transmembrane region deuterium labeled. Prior to NMR data collection, polarized infrared spectroscopy was used to establish that the peptides were reconstituted in lipid bilayers in a transmembrane helical conformation. The 2 H NMR line shapes of the three different peptides are consistent with a trimer structure formed by the designed peptide that is stabilized by interhelical hydrogen bonding of asparagines at positions 7 and 14. .

Proceedings of the National Academy of Sciences, 2005

PLoS ONE, 2014

Use of fusion protein tags to investigate lysosomal proteins can be complicated by the acidic, pr... more Use of fusion protein tags to investigate lysosomal proteins can be complicated by the acidic, protease-rich environment of the lysosome. Potential artifacts include degradation or release of the tag and acid quenching of fluorescence. Tagging can also affect protein folding, glycosylation and/or trafficking. To specifically investigate the use of fluorescent tags to reveal lysosomal localization, we tested mCherry derivatives as C-terminal tags for Niemann-Pick disease type C protein 2 (NPC2), a luminal lysosomal protein. Full-length mCherry was released from the NPC2 chimera while deletion of the 11 N-terminal residues of mCherry generated a cleavage-resistant (cr) fluorescent variant. Insertion of proline linkers between NPC2 and crmCherry had little effect while Gly-Ser linkers promoted cleavage. The NPC2-crmCherry fusion was targeted to the lysosome and restored function in NPC2-deficient cells. Fusion of crmCherry to known and candidate lysosomal proteins revealed that the linkers had different effects on lysosomal localization. Direct fusion of crmCherry impaired mannose 6phosphorylation and lysosomal targeting of the lysosomal protease tripeptidyl peptidase I (TPP1), while insertion of linkers corrected the defects. Molecular modeling suggested structural bases for the effects of different linkers on NPC2 and TPP1 fusion proteins. While mCherry fusion proteins can be useful tools for studying the lysosome and related organelles, our findings underscore the potential artifacts associated with such applications.

Nature, 2008

The M2 protein from influenza A virus is a pH-activated proton channel that mediates acidificatio... more The M2 protein from influenza A virus is a pH-activated proton channel that mediates acidification of the interior of viral particles entrapped in endosomes. M2 is the target of the anti-influenza drugs amantadine and rimantadine; recently, resistance to these drugs in humans, birds and pigs has reached more than 90% (ref. 1). Here we describe the crystal structure of the transmembrane-spanning region of the homotetrameric protein in the presence and absence of the channel-blocking drug amantadine. pH-dependent structural changes occur near a set of conserved His and Trp residues that are involved in proton gating. The drug-binding site is lined by residues that are mutated in amantadine-resistant viruses. Binding of amantadine physically occludes the pore, and might also perturb the pK(a) of the critical His residue. The structure provides a starting point for solving the problem of resistance to M2-channel blockers.

Journal of the American Chemical Society, 2008

We have developed a computational design strategy based on the R-helical coiled-coil to generate ... more We have developed a computational design strategy based on the R-helical coiled-coil to generate modular peptide motifs capable of assembling into metalloporphyrin arrays of varying lengths. The current study highlights the extension of a two-metalloporphyrin array to a four-metalloporphyrin array through the incorporation of a coiled-coil repeat unit. Molecular dynamics simulations demonstrate that the initial design evolves rapidly to a stable structure with a small rmsd compared to the original model. Biophysical characterization reveals elongated proteins of the desired length, correct cofactor stoichiometry, and cofactor specificity. The successful extension of the two-porphyrin array demonstrates how this methodology serves as a foundation to create linear assemblies of organized electrically and optically responsive cofactors.

Journal of the American Chemical Society, 2004

Protein-membrane interactions are the molecular basis of many biological processes. Among such pr... more Protein-membrane interactions are the molecular basis of many biological processes. Among such protein-membrane interactions are membrane lysis by antibiotic peptides, fertilization facilitated by fusion peptides, and viral infections mediated by fusion peptides. Other biological processes that are based on protein-membrane interactions involve proteins that are intrinsically associated with membranes such as ion channels and receptors. Therefore, development of methods for the site-specific determination of proteinmembrane interactions contributes to the understanding of the molecular basis of many such processes. The amide I band of polypeptides, which arises mainly from the amide CdO stretching vibration, has been used extensively to determine the conformation as well as orientation of transmembrane peptides in conjunction with polarized attenuated total reflection (ATR) infrared (IR) spectroscopy. 1 However, this method does not offer site specificity because the amide I band of peptides and proteins is broadened by both homogeneous and inhomogeneous mechanisms. Employing 13 CdOs may sometimes help to resolve backbone conformation of individual residues; 2 however, the local environment seen by individual side chains cannot be revealed by this approach. Here we describe a new method for studying the local environment of transmembrane peptides.

Journal of the American Chemical Society, 2006

Journal of the American Chemical Society, 2005

Metal-binding sites in metalloproteins frequently occur at the interfaces of elements of secondar... more Metal-binding sites in metalloproteins frequently occur at the interfaces of elements of secondary structure, which has enabled the retrostructural analysis of natural proteins 1 and the de novo design of helical bundles that bind diiron cofactors, 2 hemes, 3-5 porphyrins, 6 and a variety of mononuclear metal ions. 7-10 However, the design of metalloproteins containing -structure is less well developed, despite the frequent occurrence of -conformations in natural metalloproteins. Although the design of -proteins has traditionally been challenging, much progress has been made in recent years. 11 Several groups have prepared antiparallel -hairpins, 12 open-faced antiparallel -sheets, 13-16 and a toxin-like -protein featuring only a single disulfide bond. 17 Significant progress has also been made in the design of mixed R/ -folds. Here, we describe the design and molecular characterization of a -protein that mimics rubredoxin, recapitulating the binding of Fe(II/III) in a tetrathiolate geometry and allowing reversible cycling between these redox states.

Journal of Molecular Biology, 2007

We have developed an empirical residue-based potential (E z potential) for protein insertion in l... more We have developed an empirical residue-based potential (E z potential) for protein insertion in lipid membranes. Propensities for occurrence as a function of depth in the bilayer were calculated for the individual amino acid types from their distribution in known structures of helical membrane proteins. The propensities were then fit to continuous curves and converted to a potential using a reverse-Boltzman relationship. The E z potential demonstrated a good correlation with experimental data such as amino acid transfer free energy scales (water to membrane center and water to interface), and it incorporates transmembrane helices of varying composition in the membrane with trends similar to those obtained with transloconmediated insertion experiments. The potential has a variety of applications in the analysis of natural membrane proteins as well as in the design of new ones. It can help in calculating the propensity of single helices to insert in the bilayer and estimate their tilt angle with respect to the bilayer normal. It can be utilized to discriminate amphiphilic helices that assume a parallel orientation at the membrane interface, such as those of membrane-active peptides. In membrane protein design applications, the potential allows an environment-dependent selection of amino acid identities.

Journal of Molecular Biology, 2005

Membrane-spanning proteins contain both aqueous and membrane-spanning regions, both of which cont... more Membrane-spanning proteins contain both aqueous and membrane-spanning regions, both of which contribute to folding and stability. To explore the interplay between these two domains we have designed and studied the assembly of coiled-coil peptides that span from the membrane into the aqueous phase. The membrane-spanning segment is based on MS1, a transmembrane coiled coil that contains a single Asn at a buried a position of a central heptad in its sequence. This Asn has been shown to drive assembly of the monomeric peptide in a membrane environment to a mixture of dimers and trimers. The coiled coil has now been extended into the aqueous phase by addition of water-soluble helical extensions. Although too short to fold in isolation, these helical extensions were expected to interact synergistically with the transmembrane domain and modulate its stability as well as its conformational specificity for forming dimers versus trimers. One design contains Asn at a position of the aqueous helical extension, which was expected to specify a dimeric state; a second peptide, which contains Val at this position, was expected to form trimers. The thermodynamics of assembly of the hybrid peptides were studied in micelles by sedimentation equilibrium ultracentrifugation. The aqueous helical extensions indeed conferred additional stability and conformational specificity to MS1 in the expected manner. These studies highlight the delicate interplay between membrane-spanning and water-soluble regions of proteins, and demonstrate how these different environments define the thermodynamics of a given specific interaction. In this case, an Asn in the transmembrane domain provided a strong driving force for folding but failed to specify a unique oligomerization state, while an Asn in the water-soluble domain was able to define specificity for a specific aggregation state as well as modulate stability.

Journal of Molecular Biology, 2005

The driving forces behind the folding processes of integral membrane proteins after insertion int... more The driving forces behind the folding processes of integral membrane proteins after insertion into the bilayer, is currently under debate. The M2 protein from the influenza A virus is an ideal system to study lateral association of transmembrane helices. Its proton selective channel is essential for virus functioning and a target of the drug amantadine. A 25 residue transmembrane fragment of M2, M2TM, forms a four-helix bundle in vivo and in various detergents and phospholipid bilayers. Presented here are the energetic consequences for mutations made to the helix/helix interfaces of the M2TM tetramer. Analytical ultracentrifugation has been used to determine the effect of ten single-site mutations, to either alanine or phenylalanine, on the oligomeric state and the free energy of M2TM in the absence and the presence of amantadine. It was expected that many of these mutations would perturb the M2TM stability and tetrameric integrity. Interestingly, none of the mutations destabilize tetramerization. This finding suggests that M2 sacrifices stability to preserve its functions, which require rapid and specific interchange between distinct conformations involved in gating and proton conduction. Mutations might therefore restrict the full range of conformations by stabilizing a given native or non-native conformational state. In order to assess one specific conformation of the tetramer, we measured the binding of amantadine to the resting state of the channel, and examined the overall free energy of assembly of the amantadine bound tetramer. All of the mutations destabilized amantadine binding or were isoenergetic. We also find that large to small residue changes destabilize the amantadine bound tetramer whereas mutations to side-chains of similar volume stabilize this conformation. A structural model of the amantadine bound state of M2TM was generated using a novel protocol that optimizes a structure for an ensemble of neutral and disruptive mutations. The model structure is consistent with the mutational data.

Chemical Physics, 2013

Proteins involved in functions such as electron transfer or ion transport must be capable of stab... more Proteins involved in functions such as electron transfer or ion transport must be capable of stabilizing transient charged species on time scales ranging from picoseconds to microseconds. We study the influenza A M2 proton channel, containing a tryptophan residue that serves as an essential part of the proton conduction pathway. We induce a transition dipole in tryptophan by photoexcitation, and then probe the dielectric stabilization of its excited state. The magnitude of the stabilization over this time regime was larger than that generally found for tryptophan in membrane or protein environments. M2 achieves a water-like stabilization over a 25 nanosecond time scale, slower than that of bulk water, but sufficiently rapid to contribute to stabilization of charge as protons diffuse through the channel. These measurements should stimulate future MD studies to clarify the role of sidechain versus non-bulk water in defining the process of relaxation.

Chemical Physics, 2013

Using the miniprotein Trp-cage as a model, we show that D-amino acids can be used to facilitate t... more Using the miniprotein Trp-cage as a model, we show that D-amino acids can be used to facilitate the delineation of protein folding mechanism. Specifically, we study the folding-unfolding kinetics of three Trp-cage mutants where the native glycine residue near the C-terminus of the α-helix is replaced by a D-amino acid. A previous study showed that these mutations increase the Trp-cage stability, due to a terminal capping effect. Our results show that the stabilizing effect of D-asparagine and D-glutamine originates almost exclusively from a decrease in the unfolding rate, while the D-alanine mutation results in a similar decrease in the unfolding rate, but it also increases the folding rate. Together, these results support a folding mechanism wherein the α-helix formation in the transition state is nucleated at the N-terminus, whereas those long-range native interactions stabilizing this helix are developed at the downhill side of the folding free energy barrier.

Methods in enzymology, 2011

The ability to engineer novel proteins using the principles of molecular structure and energetics... more The ability to engineer novel proteins using the principles of molecular structure and energetics is a stringent test of our basic understanding of how proteins fold and maintain structure. The design of protein self-assembly has the potential to impact many fields of biology from molecular recognition to cell signaling to biomaterials. Most progress in computational design of protein self-assembly has focused on α-helical systems, exploring ways to concurrently optimize the stability and specificity of a target state. Applying these methods to collagen self-assembly is very challenging, due to fundamental differences in folding and structure of α- versus triple-helices. Here, we explore various computational methods for designing stable and specific oligomeric systems, with a focus on α-helix and collagen self-assembly.

Proteins: Structure, Function, and Bioinformatics, 2004

Cylcodextrin sugars are cyclic sugars that have a hydrophilic exterior and a hydrophobic center. ... more Cylcodextrin sugars are cyclic sugars that have a hydrophilic exterior and a hydrophobic center. This enables cyclodextrins to solubilize hydrophobic molecules in aqueous media. Cyclodextrins may inhibit aggregation by intercalating surface aromatic residues and competing with interprotein aromatic clusters (pi-pi interactions). In order to investigate this concept, the interaction of hydroxypropyl-beta-cyclodextrin (HPBCD) with melittin is studied with steady-state and time-resolved fluorescence, fluorescence polarization, circular dichroism, and IR spectroscopy. HPBCD inhibits the aggregation of melittin. This inhibition and the spectroscopic results are consistent with the lone aromatic tryptophan of the peptide being intercalated within HPBCD.

Protein science : a publication of the Protein Society, 2012

Outer membrane β-barrel proteins differ from α-helical inner membrane proteins in lipid environme... more Outer membrane β-barrel proteins differ from α-helical inner membrane proteins in lipid environment, secondary structure, and the proposed processes of folding and insertion. It is reasonable to expect that outer membrane proteins may contain primary sequence information specific for their folding and insertion behavior. In previous work, a depth-dependent insertion potential, E(z) , was derived for α-helical inner membrane proteins. We have generated an equivalent potential for TM β-barrel proteins. The similarities and differences between these two potentials provide insight into unique aspects of the folding and insertion of β-barrel membrane proteins. This potential can predict orientation within the membrane and identify functional residues involved in intermolecular interactions.

Methods in molecular biology (Clifton, N.J.), 2013

Protein-protein interactions (PPI) play central roles in biological processes, motivating us to u... more Protein-protein interactions (PPI) play central roles in biological processes, motivating us to understand the structural basis underlying affinity and specificity. In this chapter, we focus on biochemical and computational design strategies of assessing and detecting PPIs of β-barrel outer membrane proteins (OMPs). A few case studies are presented highlighting biochemical techniques used to dissect the energetics of oligomerization and determine amino acids forming the key interactions of the PPI sites. Current computational strategies for detecting/predicting PPIs are introduced, and examples of computational and rational engineering strategies applied to OMPs are presented.

Proteins: Structure, Function, and Bioinformatics, 2005

Characterizing the interactions between amino acid residues and lipid molecules is important for ... more Characterizing the interactions between amino acid residues and lipid molecules is important for understanding the assembly of transmembrane helices and for studying membrane protein folding. In this study we develop TMLIP (Trans-Membrane helix-LIPid), an empirically derived propensity of individual residue types to face lipid membrane based on statistical analysis of highresolution structures of membrane proteins. Lipid accessibilities of amino acid residues within the transmembrane (TM) region of 29 structures of helical membrane proteins are studied with a spherical probe of radius of 1.9 Å. Our results show that there are characteristic preferences for residues to face the headgroup region and the hydrocarbon core region of lipid membrane. Amino acid residues Lys, Arg, Trp, Phe, and Leu are often found exposed at the headgroup regions of the membrane, where they have high propensity to face phospholipid headgroups and glycerol backbones. In the hydrocarbon core region, the strongest preference for interacting with lipids is observed for Ile, Leu, Phe and Val. Small and polar amino acid residues are usually buried inside helical bundles and are strongly lipophobic. There is a strong correlation between various hydrophobicity scales and the propensity of a given residue to face the lipids in the hydrocarbon region of the bilayer. Our data suggest a possibly significant contribution of the lipophobic effect to the folding of membrane proteins. This study shows that membrane proteins have exceedingly apolar exteriors rather than highly polar interiors. Prediction of lipid-facing surfaces of boundary helices using TMLIP1 results in a 54% accuracy, which is significantly better than random (25% accuracy). We also compare performance of TMLIP with another lipid propensity scale, kPROT, and with several hydrophobicity scales using hydrophobic moment analysis. Proteins 2005;59:496 -509.

Proteins: Structure, Function, and Bioinformatics, 2011

The cyclic tetrapyrroles, viz. chlorophylls (Chl), their bacterial analogs bacteriochlorophylls, ... more The cyclic tetrapyrroles, viz. chlorophylls (Chl), their bacterial analogs bacteriochlorophylls, and hemes are ubiquitous cofactors of biological catalysis that are involved in a multitude of reactions. One systematic approach for understanding how Nature achieves functional diversity with only this handful of cofactors is by designing de novo simple and robust protein scaffolds with heme and/or (bacterio)chlorophyll [(B)Chls]-binding sites. This strategy is currently mostly implemented for heme-binding proteins. To gain more insight into the factors that determine heme-/(B)Chl-binding selectivity, we explored the geometric parameters of (B)Chl-binding sites in a nonredundant subset of natural (B)Chl protein structures. Comparing our analysis to the study of a nonredundant database of heme-binding helical histidines by Negron et al. (Proteins 2009;74:400-416), we found a preference for the m-rotamer in (B)Chl-binding helical histidines, in contrast to the preferred t-rotamer in heme-binding helical histidines. This may be used for the design of specific heme-or (B)Chl-binding sites in water-soluble helical bundles, because the rotamer type defines the positioning of the bound cofactor with respect to the helix interface and thus the protein-binding site. Consensus sequences for (B)Chl binding were identified by combining a computational and databasederived approach and shown to be significantly different from the consensus sequences recommended by Negron et al. (Proteins 2009;74:400-416) for heme-binding helical proteins. The insights gained in this work on helix-(B)Chls-binding pockets provide useful guidelines for the construction of reasonable (B)Chl-binding protein templates that can be optimized by computational tools. Proteins 2011; 79:463-476. V V C 2010 Wiley-Liss, Inc. Key words: protein de novo design; tetrapyrrole; histidine rotamers; ligand-binding site; consensus motif. V V C 2010 WILEY-LISS, INC. PROTEINS 463 P. Braun et al.

Proteins: Structure, Function, and Bioinformatics, 2005

In the absence of experimental structural determination, numerous methods are available to indire... more In the absence of experimental structural determination, numerous methods are available to indirectly predict or probe the structure of a target molecule. Genetic modification of a protein sequence is a powerful tool for identifying key residues involved in binding reactions or protein stability. Mutagenesis data is usually incorporated into the modeling process either through manual inspection of model compatibility with empirical data, or through the generation of geometric constraints linking sensitive residues to a binding interface. We present an approach derived from statistical studies of lattice models for introducing mutation information directly into the fitness score. The approach takes into account the phenotype of mutation (neutral or disruptive) and calculates the energy for a given structure over an ensemble of sequences. The structure prediction procedure searches for the optimal conformation where neutral sequences either have no impact or improve stability and disruptive sequences reduce stability relative to wild type. We examine three types of sequence ensembles: information from saturation mutagenesis, scanning mutagenesis, and homologous proteins. Incorporating multiple sequences into a statistical ensemble serves to energetically separate the native state and misfolded structures. As a result, the prediction of structure with a poor force field is sufficiently enhanced by mutational information to improve accuracy. Furthermore, by separating misfolded conformations from the target score, the ensemble energy serves to speed up conformational search algorithms such as Monte Carlo-based methods.

Protein Science, 2005

A peptide designed to form a homo-oligomeric transmembrane helical bundle was reconstituted into ... more A peptide designed to form a homo-oligomeric transmembrane helical bundle was reconstituted into lipid bilayers and studied by using 2 H NMR (nuclear magnetic resonance) with magic angle spinning to confirm that the helical interface corresponds to the interface intended in the design. The peptide belongs to a family of model peptides derived from a membrane-solubilized version of the water-soluble coiled-coil GCN4-P1. The variant studied here contains two asparagines thought to engage in interhelical hydrogen bonding critical to the formation of a stable trimer. For the NMR studies, three different peptides were synthesized, each with one of three consecutive leucines in the transmembrane region deuterium labeled. Prior to NMR data collection, polarized infrared spectroscopy was used to establish that the peptides were reconstituted in lipid bilayers in a transmembrane helical conformation. The 2 H NMR line shapes of the three different peptides are consistent with a trimer structure formed by the designed peptide that is stabilized by interhelical hydrogen bonding of asparagines at positions 7 and 14. .

Proceedings of the National Academy of Sciences, 2005

PLoS ONE, 2014

Use of fusion protein tags to investigate lysosomal proteins can be complicated by the acidic, pr... more Use of fusion protein tags to investigate lysosomal proteins can be complicated by the acidic, protease-rich environment of the lysosome. Potential artifacts include degradation or release of the tag and acid quenching of fluorescence. Tagging can also affect protein folding, glycosylation and/or trafficking. To specifically investigate the use of fluorescent tags to reveal lysosomal localization, we tested mCherry derivatives as C-terminal tags for Niemann-Pick disease type C protein 2 (NPC2), a luminal lysosomal protein. Full-length mCherry was released from the NPC2 chimera while deletion of the 11 N-terminal residues of mCherry generated a cleavage-resistant (cr) fluorescent variant. Insertion of proline linkers between NPC2 and crmCherry had little effect while Gly-Ser linkers promoted cleavage. The NPC2-crmCherry fusion was targeted to the lysosome and restored function in NPC2-deficient cells. Fusion of crmCherry to known and candidate lysosomal proteins revealed that the linkers had different effects on lysosomal localization. Direct fusion of crmCherry impaired mannose 6phosphorylation and lysosomal targeting of the lysosomal protease tripeptidyl peptidase I (TPP1), while insertion of linkers corrected the defects. Molecular modeling suggested structural bases for the effects of different linkers on NPC2 and TPP1 fusion proteins. While mCherry fusion proteins can be useful tools for studying the lysosome and related organelles, our findings underscore the potential artifacts associated with such applications.

Nature, 2008

The M2 protein from influenza A virus is a pH-activated proton channel that mediates acidificatio... more The M2 protein from influenza A virus is a pH-activated proton channel that mediates acidification of the interior of viral particles entrapped in endosomes. M2 is the target of the anti-influenza drugs amantadine and rimantadine; recently, resistance to these drugs in humans, birds and pigs has reached more than 90% (ref. 1). Here we describe the crystal structure of the transmembrane-spanning region of the homotetrameric protein in the presence and absence of the channel-blocking drug amantadine. pH-dependent structural changes occur near a set of conserved His and Trp residues that are involved in proton gating. The drug-binding site is lined by residues that are mutated in amantadine-resistant viruses. Binding of amantadine physically occludes the pore, and might also perturb the pK(a) of the critical His residue. The structure provides a starting point for solving the problem of resistance to M2-channel blockers.

Journal of the American Chemical Society, 2008

We have developed a computational design strategy based on the R-helical coiled-coil to generate ... more We have developed a computational design strategy based on the R-helical coiled-coil to generate modular peptide motifs capable of assembling into metalloporphyrin arrays of varying lengths. The current study highlights the extension of a two-metalloporphyrin array to a four-metalloporphyrin array through the incorporation of a coiled-coil repeat unit. Molecular dynamics simulations demonstrate that the initial design evolves rapidly to a stable structure with a small rmsd compared to the original model. Biophysical characterization reveals elongated proteins of the desired length, correct cofactor stoichiometry, and cofactor specificity. The successful extension of the two-porphyrin array demonstrates how this methodology serves as a foundation to create linear assemblies of organized electrically and optically responsive cofactors.

Journal of the American Chemical Society, 2004

Protein-membrane interactions are the molecular basis of many biological processes. Among such pr... more Protein-membrane interactions are the molecular basis of many biological processes. Among such protein-membrane interactions are membrane lysis by antibiotic peptides, fertilization facilitated by fusion peptides, and viral infections mediated by fusion peptides. Other biological processes that are based on protein-membrane interactions involve proteins that are intrinsically associated with membranes such as ion channels and receptors. Therefore, development of methods for the site-specific determination of proteinmembrane interactions contributes to the understanding of the molecular basis of many such processes. The amide I band of polypeptides, which arises mainly from the amide CdO stretching vibration, has been used extensively to determine the conformation as well as orientation of transmembrane peptides in conjunction with polarized attenuated total reflection (ATR) infrared (IR) spectroscopy. 1 However, this method does not offer site specificity because the amide I band of peptides and proteins is broadened by both homogeneous and inhomogeneous mechanisms. Employing 13 CdOs may sometimes help to resolve backbone conformation of individual residues; 2 however, the local environment seen by individual side chains cannot be revealed by this approach. Here we describe a new method for studying the local environment of transmembrane peptides.

Journal of the American Chemical Society, 2006

Journal of the American Chemical Society, 2005

Metal-binding sites in metalloproteins frequently occur at the interfaces of elements of secondar... more Metal-binding sites in metalloproteins frequently occur at the interfaces of elements of secondary structure, which has enabled the retrostructural analysis of natural proteins 1 and the de novo design of helical bundles that bind diiron cofactors, 2 hemes, 3-5 porphyrins, 6 and a variety of mononuclear metal ions. 7-10 However, the design of metalloproteins containing -structure is less well developed, despite the frequent occurrence of -conformations in natural metalloproteins. Although the design of -proteins has traditionally been challenging, much progress has been made in recent years. 11 Several groups have prepared antiparallel -hairpins, 12 open-faced antiparallel -sheets, 13-16 and a toxin-like -protein featuring only a single disulfide bond. 17 Significant progress has also been made in the design of mixed R/ -folds. Here, we describe the design and molecular characterization of a -protein that mimics rubredoxin, recapitulating the binding of Fe(II/III) in a tetrathiolate geometry and allowing reversible cycling between these redox states.

Journal of Molecular Biology, 2007

We have developed an empirical residue-based potential (E z potential) for protein insertion in l... more We have developed an empirical residue-based potential (E z potential) for protein insertion in lipid membranes. Propensities for occurrence as a function of depth in the bilayer were calculated for the individual amino acid types from their distribution in known structures of helical membrane proteins. The propensities were then fit to continuous curves and converted to a potential using a reverse-Boltzman relationship. The E z potential demonstrated a good correlation with experimental data such as amino acid transfer free energy scales (water to membrane center and water to interface), and it incorporates transmembrane helices of varying composition in the membrane with trends similar to those obtained with transloconmediated insertion experiments. The potential has a variety of applications in the analysis of natural membrane proteins as well as in the design of new ones. It can help in calculating the propensity of single helices to insert in the bilayer and estimate their tilt angle with respect to the bilayer normal. It can be utilized to discriminate amphiphilic helices that assume a parallel orientation at the membrane interface, such as those of membrane-active peptides. In membrane protein design applications, the potential allows an environment-dependent selection of amino acid identities.

Journal of Molecular Biology, 2005

Membrane-spanning proteins contain both aqueous and membrane-spanning regions, both of which cont... more Membrane-spanning proteins contain both aqueous and membrane-spanning regions, both of which contribute to folding and stability. To explore the interplay between these two domains we have designed and studied the assembly of coiled-coil peptides that span from the membrane into the aqueous phase. The membrane-spanning segment is based on MS1, a transmembrane coiled coil that contains a single Asn at a buried a position of a central heptad in its sequence. This Asn has been shown to drive assembly of the monomeric peptide in a membrane environment to a mixture of dimers and trimers. The coiled coil has now been extended into the aqueous phase by addition of water-soluble helical extensions. Although too short to fold in isolation, these helical extensions were expected to interact synergistically with the transmembrane domain and modulate its stability as well as its conformational specificity for forming dimers versus trimers. One design contains Asn at a position of the aqueous helical extension, which was expected to specify a dimeric state; a second peptide, which contains Val at this position, was expected to form trimers. The thermodynamics of assembly of the hybrid peptides were studied in micelles by sedimentation equilibrium ultracentrifugation. The aqueous helical extensions indeed conferred additional stability and conformational specificity to MS1 in the expected manner. These studies highlight the delicate interplay between membrane-spanning and water-soluble regions of proteins, and demonstrate how these different environments define the thermodynamics of a given specific interaction. In this case, an Asn in the transmembrane domain provided a strong driving force for folding but failed to specify a unique oligomerization state, while an Asn in the water-soluble domain was able to define specificity for a specific aggregation state as well as modulate stability.

Journal of Molecular Biology, 2005

The driving forces behind the folding processes of integral membrane proteins after insertion int... more The driving forces behind the folding processes of integral membrane proteins after insertion into the bilayer, is currently under debate. The M2 protein from the influenza A virus is an ideal system to study lateral association of transmembrane helices. Its proton selective channel is essential for virus functioning and a target of the drug amantadine. A 25 residue transmembrane fragment of M2, M2TM, forms a four-helix bundle in vivo and in various detergents and phospholipid bilayers. Presented here are the energetic consequences for mutations made to the helix/helix interfaces of the M2TM tetramer. Analytical ultracentrifugation has been used to determine the effect of ten single-site mutations, to either alanine or phenylalanine, on the oligomeric state and the free energy of M2TM in the absence and the presence of amantadine. It was expected that many of these mutations would perturb the M2TM stability and tetrameric integrity. Interestingly, none of the mutations destabilize tetramerization. This finding suggests that M2 sacrifices stability to preserve its functions, which require rapid and specific interchange between distinct conformations involved in gating and proton conduction. Mutations might therefore restrict the full range of conformations by stabilizing a given native or non-native conformational state. In order to assess one specific conformation of the tetramer, we measured the binding of amantadine to the resting state of the channel, and examined the overall free energy of assembly of the amantadine bound tetramer. All of the mutations destabilized amantadine binding or were isoenergetic. We also find that large to small residue changes destabilize the amantadine bound tetramer whereas mutations to side-chains of similar volume stabilize this conformation. A structural model of the amantadine bound state of M2TM was generated using a novel protocol that optimizes a structure for an ensemble of neutral and disruptive mutations. The model structure is consistent with the mutational data.

Chemical Physics, 2013

Proteins involved in functions such as electron transfer or ion transport must be capable of stab... more Proteins involved in functions such as electron transfer or ion transport must be capable of stabilizing transient charged species on time scales ranging from picoseconds to microseconds. We study the influenza A M2 proton channel, containing a tryptophan residue that serves as an essential part of the proton conduction pathway. We induce a transition dipole in tryptophan by photoexcitation, and then probe the dielectric stabilization of its excited state. The magnitude of the stabilization over this time regime was larger than that generally found for tryptophan in membrane or protein environments. M2 achieves a water-like stabilization over a 25 nanosecond time scale, slower than that of bulk water, but sufficiently rapid to contribute to stabilization of charge as protons diffuse through the channel. These measurements should stimulate future MD studies to clarify the role of sidechain versus non-bulk water in defining the process of relaxation.

Chemical Physics, 2013

Using the miniprotein Trp-cage as a model, we show that D-amino acids can be used to facilitate t... more Using the miniprotein Trp-cage as a model, we show that D-amino acids can be used to facilitate the delineation of protein folding mechanism. Specifically, we study the folding-unfolding kinetics of three Trp-cage mutants where the native glycine residue near the C-terminus of the α-helix is replaced by a D-amino acid. A previous study showed that these mutations increase the Trp-cage stability, due to a terminal capping effect. Our results show that the stabilizing effect of D-asparagine and D-glutamine originates almost exclusively from a decrease in the unfolding rate, while the D-alanine mutation results in a similar decrease in the unfolding rate, but it also increases the folding rate. Together, these results support a folding mechanism wherein the α-helix formation in the transition state is nucleated at the N-terminus, whereas those long-range native interactions stabilizing this helix are developed at the downhill side of the folding free energy barrier.