Additional Hydrophobic Residues Research Articles

Characterizing the structural and thermodynamic properties of Aβ42 and Aβ40

The self-assembly of amyloid-beta (Aβ) proteins in aqueous extracellular environments is implicated in Alzheimer's disease. Among several alloforms of Aβ proteins differing in sequence length, the 42- and 40-residue forms (Aβ42 and Aβ40) are the most abundant ones in the human body. Although the only difference is the additional I41A42 residues in the C-terminus, Aβ42 exhibits more aggregation tendency and stronger neurotoxicity than Aβ40. Here, we investigate the molecular factors that confer more aggregation potential to Aβ42 than to Aβ40 based on molecular dynamics simulations combined with solvation thermodynamic analyses. It is observed that the most salient structural feature of Aβ42 relative to Aβ40 is the more enhanced β-sheet forming tendency, in particular in the C-terminal region. While such a structural characteristic of Aβ42 will certainly serve to facilitate the formation of aggregate species rich in β-sheet structure, we also detect its interesting thermodynamic consequence. Indeed, we find from the decomposition analysis that the C-terminal region substantially increases the solvation free energy (i.e., overall “hydrophobicity”) of Aβ42, which is caused by the dehydration of the backbone moieties showing the enhanced tendency of forming the β-structure. Together with the two additional hydrophobic residues (I41A42), this leads to the higher solvation free energy of Aβ42, implying the larger water-mediated attraction toward the self-assembly. Thus, our computational results provide structural and thermodynamic grounds on why Aβ42 has more aggregation propensity than Aβ40 in aqueous environments.

YWHA/14-3-3 proteins recognize phosphorylated TFEB by a noncanonical mode for controlling TFEB cytoplasmic localization

ABSTRACTAs a master regulator of the macroautophagy/autophagy-lysosomal pathway, TFEB (transcription factor EB) plays a prominent role in regulating neurodegenerative diseases and cancer. The transcription activity of TFEB is tightly controlled by phosphorylation and dephosphorylation. Phosphorylated S211 (p-S211) of TFEB can be recognized by YWHA/14-3-3 proteins for TFEB cytoplasmic localization. Here, we characterized the interactions between phosphorylated TFEB and YWHA/14-3-3 proteins and determined the structures of YWHA/14-3-3 proteins in complex with a TFEB p-S211-peptide. Although the critical arginine for YWHA/14-3-3 recognition is missing in the N terminus of the TFEB p-S211-peptide, the C-terminal additional hydrophobic residues of the peptide unexpectedly occupy nearly half of the target-binding groove of YWHA/14-3-3 proteins, which compensates for the N-terminal defect and is distinct from the canonical YWHA/14-3-3-binding mode. Mutations of essential residues in the interaction interface between TFEB and YWHA/14-3-3 proteins disrupted their interactions and severely impaired the cytoplasmic localization of TFEB, which altered the expression of TFEB target genes and affected autophagy. Thus, YWHA/14-3-3 proteins recognize phosphorylated TFEB by a noncanonical mode for controlling TFEB cytoplasmic localization and its activity.Abbreviation: ACTB: actin beta; ALP: autophagy-lysosomal pathway; ATP6V1H: ATPase H+ transporting V1 subunit H; bHLH: basic helix-loop-helix; CLEAR: coordinated lysosomal expression and regulation; Co-IP: co-immunoprecipitation; CTSB: cathepsin B; CTSD: cathepsin D; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MITF: melanocyte inducing transcription factor; NLS: nuclear localization signal; TFEB: transcription factor EB; YWHA/14-3-3: tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein.

NoLogo: a new statistical model highlights the diversity and suggests new classes of Crm1-dependent nuclear export signals

BackgroundCrm1-dependent Nuclear Export Signals (NESs) are clusters of alternating hydrophobic and non-hydrophobic amino acid residues between 10 to 15 amino acids in length. NESs were largely thought to follow simple consensus patterns, based on which they were categorized into 6–10 classes. However, newly discovered NESs often deviate from the established consensus patterns. Thus, identifying NESs within protein sequences remains a bioinformatics challenge.ResultsWe describe a probabilistic representation of NESs using a new generative model we call NoLogo that can account for a large diversity of NESs. Using this model to predict NESs, we demonstrate improved performance over PSSM and GLAM2 models, but do not achieve the performance of the state-of-the-art NES predictor LocNES. Our findings illustrate that over 30% of NESs are best described by novel NES classes rather than the 6–10 classes proposed by current/existing models. Finally, many NESs have additional hydrophobic residues either upstream or downstream of the canonical four residues, suggesting possible functionality.ConclusionApplying the NoLogo model highlights the observation that NESs are more diverse than previously appreciated. Our work questions the practice of assigning each NES to one of several predefined NES classes. Finally, our analysis suggests a novel and testable biophysical perspective on interaction between Crm1 receptor and Crm1-dependent NESs.

Gas Sensing and Signaling in the PAS-Heme Domain of the Pseudomonas aeruginosa Aer2 Receptor.

The Aer2 chemoreceptor from Pseudomonas aeruginosa contains a PAS sensing domain that coordinates b-type heme and signals in response to the binding of O2, CO, or NO. PAS-heme structures suggest that Aer2 uniquely coordinates heme via a His residue on a 310 helix (H234 on Eη), stabilizes O2 binding via a Trp residue (W283), and signals via both W283 and an adjacent Leu residue (L264). Ligand binding may displace L264 and reorient W283 for hydrogen bonding to the ligand. Here, we clarified the mechanisms by which Aer2-PAS binds heme, regulates ligand binding, and initiates conformational signaling. H234 coordinated heme, but additional hydrophobic residues in the heme cleft were also critical for stable heme binding. O2 appeared to be the native Aer2 ligand (dissociation constant [Kd ] of 16 μM). With one exception, mutants that bound O2 could signal, whereas many mutants that bound CO could not. W283 stabilized O2 binding but not CO binding, and it was required for signal initiation; W283 mutants that could not stabilize O2 were rapidly oxidized to Fe(III). W283F was the only Trp mutant that bound O2 with wild-type affinity. The size and nature of residue 264 was important for gas binding and signaling: L264W blocked O2 binding, L264A and L264G caused O2-mediated oxidation, and L264K formed a hexacoordinate heme. Our data suggest that when O2 binds to Aer2, L264 moves concomitantly with W283 to initiate the conformational signal. The signal then propagates from the PAS domain to regulate the C-terminal HAMP and kinase control domains, ultimately modulating a cellular response.IMPORTANCEPseudomonas aeruginosa is a ubiquitous environmental bacterium and opportunistic pathogen that infects multiple body sites, including the lungs of cystic fibrosis patients. P. aeruginosa senses and responds to its environment via four chemosensory systems. Three of these systems regulate biofilm formation, twitching motility, and chemotaxis. The role of the fourth system, Che2, is unclear but has been implicated in virulence. The Che2 system contains a chemoreceptor called Aer2, which contains a PAS sensing domain that binds heme and senses oxygen. Here, we show that Aer2 uses unprecedented mechanisms to bind O2 and initiate signaling. These studies provide both the first functional corroboration of the Aer2-PAS signaling mechanism previously proposed from structure as well as a signaling model for Aer2-PAS receptors.

The N-terminus of IntDOT forms hydrophobic interactions during Holliday Junction resolution

DOT Integrase (IntDOT) is a member of the tyrosine recombinase family. It catalyzes the integration and excision reactions of an integrative and conjugative element (ICE) called CTnDOT. Like other tyrosine recombinases, the integration reaction proceeds by two sets of strand exchanges between the attDOT site on CTnDOT and an attB site in the host chromosome. The strand exchanges occur seven bases apart and define an overlap region. After the first strand exchanges a Holliday Junction (HJ) intermediate is formed. Previous work showed that a valine (V95) in a predicted alpha helix in the N-terminus of IntDOT is required for resolution of HJs to substrates and products. We have identified two additional hydrophobic residues in the helix (A92 and F99) that are involved in resolution of HJs. IntDOT proteins with substitutions at these residues form aberrant complexes in an electrophoretic mobility shift assay. We propose that these three residues participate in hydrophobic interactions that are involved in forming higher-order complexes and resolution of HJs.

A sequence-specific transcription activator motif and powerful synthetic variants that bind Mediator using a fuzzy protein interface.

Although many transcription activators contact the same set of coactivator complexes, the mechanism and specificity of these interactions have been unclear. For example, do intrinsically disordered transcription activation domains (ADs) use sequence-specific motifs, or do ADs of seemingly different sequence have common properties that encode activation function? We find that the central activation domain (cAD) of the yeast activator Gcn4 functions through a short, conserved sequence-specific motif. Optimizing the residues surrounding this short motif by inserting additional hydrophobic residues creates very powerful ADs that bind the Mediator subunit Gal11/Med15 with high affinity via a "fuzzy" protein interface. In contrast to Gcn4, the activity of these synthetic ADs is not strongly dependent on any one residue of the AD, and this redundancy is similar to that of some natural ADs in which few if any sequence-specific residues have been identified. The additional hydrophobic residues in the synthetic ADs likely allow multiple faces of the AD helix to interact with the Gal11 activator-binding domain, effectively forming a fuzzier interface than that of the wild-type cAD.

The coexistence of an equal amount of Alzheimer's amyloid‐β 40 and 42 forms structurally stable and toxic oligomers through a distinct pathway

Fibrillar amyloid-β (Aβ) is the major constituent of senile plaques in the brain of patients with Alzheimer's disease (AD). Aβ is a short peptide generated from amyloid precursor protein with two main isoforms, Aβ40 and Aβ42, with the latter having two additional hydrophobic residues at the C-terminus. The two isoforms have distinct characteristics, in which Aβ42 plays a more pathogenic role. Some early-onset familial AD cases possess an elevated Aβ42/Aβ40 level, and biochemical studies show the two species interact with each other. Therefore, understanding structural conversion in the aggregation of mixed Aβ isoforms is essential for elucidating AD pathogenesis. Here, we systematically examined the differences between Aβ42, Aβ40 and various Aβ42/Aβ40 mixtures by monitoring the fibrillization kinetics, epitope changes, assembly, morphology and induced cytotoxicity. We found that the minor Aβ species in different mixing ratios modulated the major aggregation pathway. Size exclusion chromatography, circular dichroism spectroscopy and photo-crosslinking assay showed that soluble Aβ42 oligomers were stabilized after Aβ40 addition, and the equimolar Aβ42/Aβ40 mixture rapidly formed spherical oligomers. These oligomers were the most toxic among those examined as evidenced by neurite degeneration and neuronal toxicity. However, the oligomers were not responsible for intracellular calcium elevation. Overall, our results demonstrated that differently mixed Aβ species repartitioned oligomer intermediates on the major aggregation pathway. Furthermore, the equimolar mixture rapidly formed structurally stable and the most toxic oligomers. These results provided information on the potential pathological mechanisms underlying the elevated Aβ42/Aβ40 ratio in familial AD patients and in the local environment of sporadic AD brains.

A Mutation in the Ebola Virus Envelope Glycoprotein Restricts Viral Entry in a Host Species- and Cell-Type-Specific Manner

Zaire Ebola virus (EBOV) is a zoonotic pathogen that causes severe hemorrhagic fever in humans. A single viral glycoprotein (GP) mediates viral attachment and entry. Here, virus-like particle (VLP)-based entry assays demonstrate that a GP mutant, GP-F88A, which is defective for entry into a variety of human cell types, including antigen-presenting cells (APCs), such as macrophages and dendritic cells, can mediate viral entry into mouse CD11b(+) APCs. Like that of wild-type GP (GP-wt), GP-F88A-mediated entry occurs via a macropinocytosis-related pathway and requires endosomal cysteine proteases and an intact fusion peptide. Several additional hydrophobic residues lie in close proximity to GP-F88, including L111, I113, L122, and F225. GP mutants in which these residues are mutated to alanine displayed preferential and often impaired entry into several cell types, although not in a species-specific manner. Niemann-Pick C1 (NPC1) protein is an essential filovirus receptor that binds directly to GP. Overexpression of NPC1 was recently demonstrated to rescue GP-F88A-mediated entry. A quantitative enzyme-linked immunosorbent assay (ELISA) demonstrated that while the F88A mutation impairs GP binding to human NPC1 by 10-fold, it has little impact on GP binding to mouse NPC1. Interestingly, not all mouse macrophage cell lines permit GP-F88A entry. The IC-21 cell line was permissive, whereas RAW 264.7 cells were not. Quantitative reverse transcription (RT)-PCR assays demonstrate higher NPC1 levels in GP-F88A permissive IC-21 cells and mouse peritoneal macrophages than in RAW 264.7 cells. Cumulatively, these studies suggest an important role for NPC1 in the differential entry of GP-F88A into mouse versus human APCs.

At the Source: Treating Heart Failure by Altering Muscle Motor Function

### Cardiac Myosin Activation: A Potential Therapeutic Approach for Systolic Heart Failure Malik et al Science . 2011;331:1439–1443. A new study in Science provides proof of the principle for directly modulating the cardiac muscle's motor, myosin, as a therapeutic target in heart failure. The human and economic tolls from heart failure (HF) are significant in both developed and undeveloped countries.1–3 Although HF can take many forms,4,5 it is most commonly associated with decreased cardiac contractility and is, therefore, referred to as systolic HF (sHF).6 Current therapeutic strategies rely on blocking neurohormonal activation by inhibiting associated pathways or by receptor blockade using either β-adrenergic or aldosterone blockers.7,8 Alternatively, some therapies are directed at increasing cardiac contractility in an effort to restore sufficient blood flow, usually by activating second messenger signaling pathways that increase cardiomyocyte calcium levels and augment sarcomere contraction.9 Both strategies suffer from a lack of specificity in that they target proteins upstream of the actual contractile events that underlie force production. Affecting important signaling pathways or receptor-ligand interactions can impact cellular function and perturb homeostasis in ways unrelated to the contractile apparatus, leading to undesirable side effects, such as hypotension, tachycardia, and even arrhythmias that can lead to sudden death.10 Recognizing the significant limitations of current therapeutic modalities, Malik and co-workers have directed their efforts at directly modulating cardiac contractility by tuning motor function at the source, the myosin heavy chain. The cardiac myosin heavy chains are encoded by two genes, MYH6 , which encodes α myosin heavy chain, and MYH7 , which encodes β myosin heavy chain, the latter being the predominant isoform in human ventricle. Although having greater than 90% amino acid homology, the two proteins differ significantly at the biochemical level. In comparison with α myosin, β myosin is relatively more efficient in force production as a function of energy consumption, with …

Amyloid β-Protein Monomer Folding: Free-Energy Surfaces Reveal Alloform-Specific Differences

Alloform-specific differences in structural dynamics between amyloid β-protein (Aβ) 40 and Aβ42 appear to underlie the pathogenesis of Alzheimer's disease. To elucidate these differences, we performed microsecond timescale replica-exchange molecular dynamics simulations to sample the conformational space of the Aβ monomer and constructed its free-energy surface. We find that neither peptide monomer is unstructured, but rather that each may be described as a unique statistical coil in which five relatively independent folding units exist, comprising residues 1–5, 10–13, 17–22, 28–37, and 39–42, which are connected by four turn structures. The free-energy surfaces of both peptides are characterized by two large basins, comprising conformers with either substantial α-helix or β-sheet content. Conformational transitions within and between these basins are rapid. The two additional hydrophobic residues at the Aβ42 C-terminus, Ile41 and Ala42, significantly increase contacts within the C-terminus, and between the C-terminus and the central hydrophobic cluster (Leu17-Ala21). As a result, the β-structure of Aβ42 is more stable than that of Aβ40, and the conformational equilibrium in Aβ42 shifts towards β-structure. These results suggest that drugs stabilizing α-helical Aβ conformers (or destabilizing the β-sheet state) would block formation of neurotoxic oligomers. The atomic-resolution conformer structures determined in our simulations may serve as useful targets for this purpose. The conformers also provide starting points for simulations of Aβ oligomerization—a process postulated to be the key pathogenetic event in Alzheimer's disease.

Modification of the Thermal Unfolding Pathways of Myoglobin upon Drug Interaction in Different Aqueous Media

In this work, we have analyzed the influence of two structurally related phenothiazine drugs, promazine and triflupromazine hydrochlorides, when bound to myoglobin, a model protein, and how the drug concentration and solution conditions may affect the denaturation process of this protein. In this manner, we derive the thermodynamic quantities of the unfolding process by using a spectroscopic technique such as UV-vis spectroscopy at different drugs concentrations and at pH 2.5, 5.5, and 9.0. To do this, a thermodynamic model was used which included experimental data corresponding to the pre- and post-transition into the observable transition. It has been found that both drugs play a destabilizing role for the protein, at least at low concentrations. In addition, at acidic pH and higher drug concentrations, a stabilizing effect can be observed, which may be related to the formation of some type of protein refolding, subsequent aggregation, or both. The reason for this behavior has been suggested to be the different protein conformations at acidic pH, the increase of solvent-exposed hydrophobic and hydrophilic residues after denaturation and/or binding, and the different strength of drug-protein interactions when changing the solution conditions. For this reason, thermodynamic quantities such as Gibbs energies, DeltaG, and entropies of unfolding, DeltaS(m), increase as the solution pH increases provided that additional solvent-exposed hydrophobic residues are present, which were previously buried at room temperature. Moreover, the larger binding affinity at pH 9.0 due to enhanced electrostatic interactions between protein and drug molecules (drug and protein differ in their net electrical charge) additionally collaborates to this residue exposition to solvent as a consequence of the alteration of protein conformation as due to drug binding. Comparison of thermodynamic data between promazine and triflupromazine hydrochlorides also shows that drug-protein affinity and hydrophobicity also affect the thermodynamic denaturation parameters.

Structural and Functional Basis for (S)-Allantoin Formation in the Ureide Pathway

The ureide pathway, which mediates the oxidative degradation of uric acid to (S)-allantoin, represents the late stage of purine catabolism in most organisms. The details of uric acid metabolism remained elusive until the complete pathway involving three enzymes was recently identified and characterized. However, the molecular details of the exclusive production of one enantiomer of allantoin in this pathway are still undefined. Here we report the crystal structure of 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) decarboxylase, which catalyzes the last reaction of the pathway, in a complex with the product, (S)-allantoin, at 2.5-A resolution. The homodimeric helical protein represents a novel structural motif and reveals that the active site in each monomer contains no cofactors, distinguishing this enzyme mechanistically from other cofactor-dependent decarboxylases. On the basis of structural analysis, along with site-directed mutagenesis, a mechanism for the enzyme is proposed in which a decarboxylation reaction occurs directly, and the invariant histidine residue in the OHCU decarboxylase family plays an essential role in producing (S)-allantoin through a proton transfer from the hydroxyl group at C4 to C5 at the re-face of OHCU. These results provide molecular details that address a longstanding question of how living organisms selectively produce (S)-allantoin.

Docking Interactions Induce Exposure of Activation Loop in the MAP Kinase ERK2

MAP kinases bind activating kinases, phosphatases, and substrates through docking interactions. Here, we report a 1.9 A crystallographic analysis of inactive ERK2 bound to a "D motif" docking peptide (pepHePTP) derived from hematopoietic tyrosine phosphatase, a negative regulator of ERK2. In this complex, the complete D motif interaction defined by mutagenic analysis is observed, including extensive electrostatic interactions with the "CD" site of the kinase. Large conformational changes occur in the activation loop where the dual phosphorylation sites, which are buried in the inactive form of ERK2, become exposed to solvent in the complex. Similar conformational changes occur in a complex between ERK2 and a MEK2 (MAP/ERK kinase-2)-derived D motif peptide (pepMEK2). D motif peptides are known to bind homologous loci in the MAP kinases p38alpha and JNK1, also inducing conformational changes in these enzymes. However, the binding interactions and conformational changes are unique to each, thus contributing to specificity among MAP kinases.

Structural Basis for the High-affinity Binding of Nucleoporin Nup1p to the Saccharomyces cerevisiae Importin-β Homologue, Kap95p

Macromolecules are transported across the nuclear envelope most frequently by karyopherin/importin-beta superfamily members that are constructed from HEAT repeats. Transport of Kap95p (yeast importin-beta), the principal carrier for protein import, through nuclear pore complexes is facilitated by interactions with nucleoporins containing FG repeats. However, Nup1p interacts more strongly with Kap95p than other FG-nucleoporins. To establish the basis of this increased affinity, we determined the structure of Kap95p complexed with Nup1p residues 963-1076 that contain the high-affinity Kap95p binding site. Nup1p binds Kap95p at three sites between the outer A-helices of HEAT repeats 5, 6, 7 and 8. At each site, phenylalanine residues from Nup1p are buried in hydrophobic depressions between adjacent HEAT repeats. Although the Nup1p and generic FG-nucleoporin binding sites on Kap95p overlap, Nup1p binding differs markedly and has contributions from additional hydrophobic residues, together with interactions generated by the intimate contact of the linker between Nup1 residues 977-987 with Kap95p. The length and composition of this linker is crucial and suggests how differences in affinity for Kap95p both between and within FG-nucleoporins arise.

Characterization of the Endosomal Sorting Signal of the Cation-dependent Mannose 6-Phosphate Receptor

Intracellular cycling of the cation-dependent mannose 6-phosphate receptor (CD-MPR) between different compartments is directed by signals localized in its cytoplasmic tail. A di-aromatic motif (Phe18-Trp19 with Trp19 as the key residue) in its cytoplasmic tail is required for the sorting of the receptor from late endosomes back to the Golgi apparatus. However, the cation-independent mannose 6-phosphate receptor (CI-MPR) lacks such a di-aromatic motif. Therefore the ability of amino acids other than aromatic residues to replace Trp19 in the CD-MPR cytoplasmic tail was tested. Mutant constructs with bulky hydrophobic residues (valine, isoleucine, or leucine) instead of Trp19 exhibited 30-60% decreases in binding to the tail interacting protein of 47 kDa (Tip47), a protein mediating this transport step, and partially prevented receptor delivery to lysosomes. Decreasing hydrophobicity of residues at position 19 resulted in further impairment of Tip47 binding and an increase of receptor accumulation in lysosomes. Intriguingly, mutants mislocalized to lysosomes did not completely co-localize with a lysosomal membrane protein, which might suggest the presence of subdomains within lysosomes. These data indicate that sorting of the CD-MPR in late endosomes requires a distinct di-aromatic motif with only limited possibilities for variations, in contrast to the CI-MPR, which seems to require a putative loop (Pro49-Pro-Ala-Pro-Arg-Pro-Gly55) along with additional hydrophobic residues in the cytoplasmic tail. This raises the possibility of two separate binding sites on Tip47 because both receptors require binding to Tip47 for endosomal sorting.

A Structure-based Mutational Analysis of Cyclophilin 40 Identifies Key Residues in the Core Tetratricopeptide Repeat Domain That Mediate Binding to Hsp90

Cyclophilin 40 (CyP40) is a tetratricopeptide repeat (TPR)-containing immunophilin and a modulator of steroid receptor function through its binding to heat shock protein 90 (Hsp90). Critical to this binding are the carboxyl-terminal MEEVD motif of Hsp90 and the TPR domain of CyP40. Two different models of the CyP40-MEEVD peptide interaction were used as the basis for a comprehensive mutational analysis of the Hsp90-interacting domain of CyP40. Using a carboxyl-terminal CyP40 construct as template, 24 amino acids from the TPR and flanking acidic and basic domains were individually mutated by site-directed mutagenesis, and the mutants were coexpressed in yeast with a carboxyl-terminal Hsp90beta construct and qualitatively assessed for binding using a beta-galactosidase filter assay. For quantitative assessment, mutants were expressed as glutathione S-transferase fusion proteins and assayed for binding to carboxyl-terminal Hsp90beta using conventional pulldown and enzyme-linked immunosorbent assay microtiter plate assays. Collectively, the models predict that the following TPR residues help define a binding groove for the MEEVD peptide: Lys-227, Asn-231, Phe-234, Ser-274, Asn-278, Lys-308, and Arg-312. Mutational analysis identified five of these residues (Lys-227, Asn-231, Asn-278, Lys-308, and Arg-312) as essential for Hsp90 binding. The other two residues (Phe-234 and Ser-274) and another three TPR domain residues not definitively associated with the binding groove (Leu-284, Lys-285, and Asp-329) are required for efficient Hsp90 binding. These data confirm the critical importance of the MEEVD binding groove in CyP40 for Hsp90 recognition and reveal that additional charged and hydrophobic residues within the CyP40 TPR domain are required for Hsp90 binding.

Achieving stability and conformational specificity in designed proteins via binary patterning

We have developed a method to determine the optimal binary pattern (arrangement of hydrophobic and polar amino acids) of a target protein fold prior to amino acid sequence selection in protein design studies. A solvent accessible surface is generated for a target fold using its backbone coordinates and “generic” side-chains, which are constructs whose size and shape are similar to an average amino acid. Each position is classified as hydrophobic or polar according to the solvent exposure of its generic side-chain. The method was tested by analyzing a set of proteins in the Protein Data Bank and by experimentally constructing and analyzing a set of engrailed homeodomain variants whose binary patterns were systematically varied. Selection of the optimal binary pattern results in a designed protein that is monomeric, well-folded, and hyperthermophilic. Homeodomain variants with fewer hydrophobic residues are destabilized, while additional hydrophobic residues induce aggregation. Binary patterning, in conjunction with a force field that models folded state energies, appears sufficient to satisfy two basic goals of protein design: stability and conformational specificity.

Tolerance of a protein to multiple polar-to-hydrophobic surface substitutions.

Hydrophobic substitutions at solvent-exposed positions in two alpha-helical regions of the bacteriophage P22 Arc repressor were introduced by combinatorial mutagenesis. In helix A, hydrophobic residues were tolerated individually at each of the five positions examined, but multiple substitutions were poorly tolerated as shown by the finding that mutants with more than two additional hydrophobic residues were biologically inactive. Several inactive helix A variants were purified and found to have reduced thermal stability relative to wild-type Arc, with a rough correlation between the number of polar-to-hydrophobic substitutions and the magnitude of the stability defect. Quite different results were obtained in helix B, where variants with as many as five polar-to-hydrophobic substitutions were found to be biologically active and one variant with three hydrophobic substitutions had a t(m) 6 degrees C higher than wild-type. By contrast, a helix A mutant with three similar polar-to-hydrophobic substitutions was 23 degrees C less stable than wild-type. Also, one set of three polar-to-hydrophobic substitutions in helix B was tolerated when introduced into the wild-type background but not when introduced into an equally active mutant having a nearly identical structure. Context effects occur both when comparing different regions of the same protein and when comparing the same region in two different homologues.

Specific interactions of bovine and human β-casomorphin-7 with Cu(II) ions

Complex formation between Cu(II) and human and bovine β-casomorphin heptapeptides, Tyr-Pro-Phe-Val-Glu-Pro-Ile and Tyr-Pro-Phe-Pro-Gly-Pro-Ile, respectively, was investigated by pH potentiometry and spectroscopic (CD, EPR and electronic absorption) techniques. The results showed the critical impact of Pro residues on the complex equilibria formed. The presence of the Pro residue at the second position leads to formation of very stable dimeric species in which two metal ions co-ordinate to N-terminal {NH 2,CO} binding sites of one peptide molecule and the deprotonated phenolic oxygen of the second ligand molecule. The presence of two additional hydrophobic residues on the C-terminal makes heptapeptide molecule much more effective ligand than its pentapeptide N-terminal fragment.

The Effect of Cytoplasmic Domain Mutations on Membrane Anchoring and Glycoprotein Processing of Herpes Simplex Virus Type 1 Glycoprotein C

The predicted amino acid sequence of herpes simplex virus type 1 glycoprotein C (HSV-1 gC) shows that it has the features of a typical type 1 integral membrane protein: a cleavable N-terminal signal sequence, a glycosylated ectodomain, a single transmembrane domain, and a small, charged cytoplasmic domain. In an earlier investigation of the function of the gC cytoplasmic domain, it was shown that the gC synthesized by a gC mutant, d12, which lacked the last three residues of the transmembrane domain and the entire cytoplasmic domain, was initially synthesized as a membrane bound glycoprotein, but was not stably anchored in the plasma membrane. In this study, we generated a panel of four HSV-1 gC mutants with novel cytoplasmic domains in order to further delineate the role of this domain in stable anchoring and to investigate the role of charged residues in this process. The cytoplasmic domain mutants produced significant quantities of a novel precursor (pgC-86K), approximately 6K smaller than the wild-type gC precursor. The quantity of pgC-86K correlated with the number of positive charges in the cytoplasmic domain. Although the nature of the novel form of the precursor is unclear, its correlation with cytoplasmic domain charge suggests that an important function of this domain is to influence gC processing. Significantly, restoration of the carboxy-terminal 3 amino acids of the gC transmembrane domain restored the wild-type anchoring phenotype to gC, indicating that the cytoplasmic domain is not required for membrane anchoring. Further characterization of d12 gC confirmed that this glycoprotein is not released from the membrane by proteolysis. We suggest that the addition of three additional hydrophobic residues to the d12 transmembrane domain increases its hydrophobicity enough to stabilize membrane anchoring of the glycoprotein.