Solvent accessibility, protein surfaces, and protein folding.

Studies of the native structures of proteins, together with measurements of the thermodynamic properties of the transition between unfolded and native states, have defined the major components of the forces that stabilize native protein structures. However, the nature of the intermediates in the folding process remains largely hypothetical. It is a fairly widespread and not implausible assumption that the intermediates in the folding of a monomeric protein contain the same kinds of secondary and tertiary structures that appear in the native conformation, and that, although unstable, their lifetimes are prolonged by forces similar to those that stabilize the native structure. We wished to examine what happens if, during the folding of a monomeric protein, regions of secondary structure come together to form an intermediate of reduced instability. We applied calculations of accessible surface area (a measure of hydrophobic stabilization) and parameterized nonbonded energy calculations (measuring the strengths of van der Waals forces) to identify the kinds of stabilizing interactions that might be available to such an intermediate. First, we analyzed the total buried surface area of two types of proteins into contributions from formation of secondary structure alone, interaction of pairs of secondary-structural elements, the formation of the structure alone, interaction of pairs of secondary-structural elements, the formation of the complete secondary structure without the turns, and the complete native structure. The formation of secondary structure alone, without tertiary-structural interactions, buries roughly half the surface that the complete structure does. We then analyzed in more detail the approach of two alpha-helices to form a complex, as an illustrative example of the nature of the interaction between compact structural units which remain fairly rigid during their interaction. Many features of the results are not limited to the interaction of alpha-helices. (The results therefore neither confirm nor refute the hypothesis that alpha-helices are intermediates in the folding proteins). We find that the first forces to be felt upon approach arise from solvent conditions on the relative position and orientation of the two helices as does the close packing which optimizes the van der Waals interactions at shorter distances apart. Therefore there appears to be a range of distances in which hydrophobic interactions could create a nonspecific complex between two helices in which the side chains might have sufficient time to seek the proper interdigitation observed in the native structure, where the two helices are in intimate contact. Indeed, we find that only in the final stages of approach is the native geometry the most stable; in the region in which solvent-exclusion forces predominate, the conformation with helix axes parallel is more stable than the native conformation, in the cases we examined...     

Microtubule-associated protein, MAP2, is a calcium-binding protein

Calcium has been suggested to be an important element in the regulation of microtubule dynamics 'in vivo'. In this report we have analyzed the possibility that microtubule-associated protein 2 (MAP2) binds calcium. MAP2 was blue-stained with the cationic carbocyanine dye 'stains-all' in a similar way to that of calcium-binding proteins and bound 45Ca as estimated from dot-blotting experiments. The calcium-binding characteristics of MAP2, determined by equilibrium dialysis, indicated that MAP2 bound about 3 mol (n = 2.9 +/- 0.4) of calcium per mol of protein (Kd = (0.9 +/- 0.2).10(-5) M). Analysis of the Scatchard plots from equilibrium dialysis and dot-blot assays indicated that MAP2 also presented low-affinity calcium-binding sites (Kd = (0.3 +/- 0.2).10(-4) M). Incubation of nitrocellulose blots of proteolytically digested MAP2 with 45Ca indicated that the calcium-binding sites were located in the region that is not involved in the interaction with tubulin (projection region).     

Cockroach larval-specific protein, a tyrosine-rich serum protein

Larval-specific protein (LSP) is the most abundant protein in the hemolymph of cockroaches shortly before molting, but is rapidly cleared from the hemolymph during the molt (Kunkel, J. G., and Lawler, D. M. (1974) Comp. Biochem. Physiol. 47B, 697-710). Blatta orientalis LSP was purified by sedimentation in preparative sucrose gradients followed by 2-hydroxypropylamino-cellulose anion-exchange chromatography and gel filtration on a column of Bio-Gel A-1.5m. The amino acid composition of LSP includes 16.3 mol % tyrosine and 4.9 mol % phenylalanine, but virtually no cysteine and little methionine. The following physical properties were determined for LSP: R8 = 68.3 A, 8(20),w = 17.8, and V = 0.723. From these values an Mr = 507,900 was calculated. In electron micrographs, LSP appears as rectangular particles of 121 by 134 A. In disc polyacrylamide gel electrophoresis, native LSP exhibits a single band, but in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, LSP is resolved into a doublet of closely spaced bands of Mr = 88,100 and 84,400 present in a ratio of 1.38:1. These data indicate that native B. orientalis LSP is a hexamer of subunits averaging approximately Mr = 86,000. Crossed immunoelectrophoresis of Blattella germanica larval serum indicates that LSP in that species is a hexamer composed of a random assortment of two subunits of different charge in the ratio 1.25:1. The amino acid composition and physical properties of LSP suggest that LSP may be the hemimetabolous analogue of the tyrosine- and phenylalanine-rich storage proteins of holometabolous insects.     

Geometry, thermodynamics, and protein

We derive a new continuous free energy formula for protein folding. We obtain the formula first by adding hydrophobic effect to a classical free energy formula for cavities in water. We then obtain the same formula by geometrically pursuing the structure that fits best the well-known global geometric features of native structures of globular proteins: 1. high density; 2. small surface area; 3. hydrophobic core; 4. forming domains for long polypeptide chains. Conformations of a protein are presented as an all atom CPK model where each atom is a ball B(x_i,r_i). All conformations satisfy generally defined steric conditions. For each conformation P of a globular protein, there is a closed thermodynamic system Ω_P⊃P bounded by the molecular surface M_P. Both methods derive the same free energy aV(P)+bA(P)+cW(P), where a,b,c>0, V(P), A(P), and W(P) are volume of Ω_P, area of M_P, and area of the hydrophobic surface W_P⊂M_P, which quantifies hydrophobic effect.Minimizing W(P) is sufficient to produce statistically significant native like secondary structures and hydrogen bonds in the proteins we simulated.     

Metal affinity protein precipitation: effects of mixing, protein concentration, and modifiers on protein fractionation

Previous work by us and others has shown that mixing impacts apparent protein solubility in single protein precipitations. In this work, we probe the effects of contacting conditions on fractional precipitation behavior at the bench scale. We have chosen metal affinity precipitation as our model system; the kinetics of this mode of precipitation are very rapid and largely irreversible and, consequently, mixing conditions govern the extent of fractionation and purity of the product in such a process. Our experimental strategy involved a three-pronged approach to control the effects contacting conditions on precipitate yield, purity, and particle size distribution. First, we studied the impact of process variables that control precipitant concentrations in the reactor including impeller speed and precipitant addition rate. Second, we controlled the rate of precipitation by changing the initial protein concentration to alter the protein-protein collision rate. Third, we examined the role of the molecular-level kinetics of affinity precipitation by using modifiers that compete with surface moieties to bind the metal ion, thereby reducing its availability. Our model process and protein system consisted of zinc precipitations of mixtures of bovine serum albumin and bovine gamma-globulins, carried out at a nominal 1-L scale; glycine was examined as a modifier. Faster impeller speeds and lower precipitant addition rates increased the desired protein yields, decreased purities, and reduced average precipitate particle size. Higher initial protein concentrations were found to produce precipitates with higher yields, lower purities and diminished particle size. Experiments with glycine indicated that modifiers in the precipitant solution serve to increase product purity, decrease yield, and increase the average particle size in bench-scale precipitations. (c) 1995 John Wiley & Sons, Inc.     

The interface of protein structure, protein biophysics, and molecular evolution

Abstract The interface of protein structural biology, protein biophysics, molecular evolution, and molecular population genetics forms the foundations for a mechanistic understanding of many aspects of protein biochemistry. Current efforts in interdisciplinary protein modeling are in their infancy and the state-of-the art of such models is described. Beyond the relationship between amino acid substitution and static protein structure, protein function, and corresponding organismal fitness, other considerations are also discussed. More complex mutational processes such as insertion and deletion and domain rearrangements and even circular permutations should be evaluated. The role of intrinsically disordered proteins is still controversial, but may be increasingly important to consider. Protein geometry and protein dynamics as a deviation from static considerations of protein structure are also important. Protein expression level is known to be a major determinant of evolutionary rate and several considerations including selection at the mRNA level and the role of interaction specificity are discussed. Lastly, the relationship between modeling and needed high-throughput experimental data as well as experimental examination of protein evolution using ancestral sequence resurrection and in vitro biochemistry are presented, towards an aim of ultimately generating better models for biological inference and prediction.     

XtalView, protein structure solution and protein graphics, a short history

From a user's point-of-view we are in the Golden Age of protein crystallographic software. In the past few decades, solving protein structures has gone from a task requiring man-months of effort to a process requiring minutes on an ordinary laptop with no human intervention required. The birth of XtalView coincided with the mainstream use of synchrotron radiation, seleno-Met phasing and it continues to be used in this age of robotic crystallization, Fed-Ex data collection and fully automated structure solution "pipelines". This article is a retrospective history of protein crystallographic computing and a discussion of the current state of the art.     

WD-repeat-propeller-FYVE protein, ProF, binds VAMP2 and protein kinase Czeta

We have recently identified a protein, consisting of seven WD repeats, presumably forming a beta-propeller, and a domain identified in Fab1p, YOTB, VAC1p, and EEA1 (FYVE) domain, ProF. The FYVE domain targets the protein to vesicular membranes, while the WD repeats allow binding of the activated kinases Akt and protein kinase (PK)Czeta. Here, we describe the vesicle-associated membrane protein 2 (VAMP2) as interaction partner of ProF. The interaction is demonstrated with overexpressed and endogenous proteins in mammalian cells. ProF and VAMP2 partially colocalize on vesicular structures with PKCzeta and the proteins form a ternary complex. VAMP2 can be phosphorylated by activated PKCzeta in vitro and the presence of ProF increases the PKCzeta-dependent phosphorylation of VAMP2 in vitro. ProF is an adaptor protein that brings together a kinase with its substrate. VAMP2 is known to regulate docking and fusion of vesicles and to play a role in targeting vesicles to the plasma membrane. The complex may be involved in vesicle cycling in various secretory pathways.     

Fyn binding protein, Fyb, interacts with mammalian actin binding protein, mAbp1

The immune cell specific protein Fyn-T binding protein (Fyb) has been identified as a target of the Yersinia antiphagocytic effector Yersinia outer protein H (YopH), but its role in macrophages is unknown. By using Fyb domains as bait to screen a mouse lymphoma cDNA library, we identified a novel interaction partner, mammalian actin binding protein 1 (mAbp1). We show that mAbp1 binds the Fyb N-terminal via its C-terminally located src homology 3 domain. The interaction between Fyb and mAbp1 is detected in macrophage lysates and the proteins co-localize with F-actin in the leading edge. Hence, mAbp1 is likely to constitute a downstream effector of Fyb involved in F-actin dynamics.     

Differential IgG-binding characteristics of staphylococcal protein A, streptococcal protein G, and a chimeric protein AG

Various Gram-positive bacteria express different types of IgG-binding receptors, each of which displaying certain unique binding properties. To evaluate specificity and avidity aspects of the differential binding pattern, a set of competitive binding assays was employed, by using staphylococcal protein A (SPA), streptococcal protein G (SPG), and a chimeric protein AG. These receptors were analyzed, in a reciprocal fashion, for binding and inhibition of binding to a selected panel of polyclonal and monoclonal Ig. Results of the study reveal that a majority of the determinants on human and bovine IgG, recognized by SPA and SPG, are either coextensive or closely overlapping. Accordingly, a minor portion of the determinants appear to be unique in the sense that a particular determinant(s) is selectively identified by one of the two receptors. Binding assays involving purified Fc fragments from human IgG, suggest that SPG shows exclusive specificity for an Fab region determinant(s) not recognized by SPA, whereas the Fc determinants for SPA and SPG are identical or overlapping. Furthermore, one of the IgG subclasses of bovine origin appears to be seen by the SPG receptor only. The competition study also demonstrates that the novel chimeric protein AG receptor shows higher or equal avidity for variants of human IgG molecules compared to the best of its parental constituents. It can thus be deduced that chimeric receptors might be useful as optimized tools for immunologic applications.     

Phosphorylation of connexin 32, a hepatocyte gap-junction protein, by cAMP-dependent protein kinase, protein kinase C and Ca2+/calmodulin-dependent protein kinase II

Phosphorylation of connexin 32, the major liver gap-junction protein, was studied in purified liver gap junctions and in hepatocytes. In isolated gap junctions, connexin 32 was phosphorylated by cAMP-dependent protein kinase (cAMP-PK), by protein kinase C (PKC) and by Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM-PK II). Connexin 26 was not phosphorylated by these three protein kinases. Phosphopeptide mapping of connexin 32 demonstrated that cAMP-PK and PKC primarily phosphorylated a seryl residue in a peptide termed peptide 1. PKC also phosphorylated seryl residues in additional peptides. CA2+/CaM-PK II phosphorylated serine and to a lesser extent, threonine, at sites different from those phosphorylated by the other two protein kinases. A synthetic peptide PSRKGSGFGHRL-amine (residues 228-239 based on the deduced amino acid sequence of rat connexin 32) was phosphorylated by cAMP-PK and by PKC, with kinetic properties being similar to those for other physiological substrates phosphorylated by these enzymes. Ca2+/CaM-PK II did not phosphorylate the peptide. Phosphopeptide mapping and amino acid sequencing of the phosphorylated synthetic peptide indicated that Ser233 of connexin 32 was present in peptide 1 and was phosphorylated by cAMP-PK or by PKC. In hepatocytes labeled with [32P]orthophosphoric acid, treatment with forskolin or 20-deoxy-20-oxophorbol 12,13-dibutyrate (PDBt) resulted in increased 32P-incorporation into connexin 32. Phosphopeptide mapping and phosphoamino acid analysis showed that a seryl residue in peptide 1 was most prominently phosphorylated under basal conditions. Treatment with forskolin or PDBt stimulated the phosphorylation of peptide 1. PDBt treatment also increased the phosphorylation of seryl residues in several other peptides. PDBt did not affect the cAMP-PK activity in hepatocytes. It has previously been shown that phorbol ester reduces dye coupling in several cell types, however in rat hepatocytes, dye coupling was not reduced by treatment with PDBt. Thus, activation of PKC may have differential effects on junctional permeability in different cell types; one source of this variability may be differences in the sites of phosphorylation in different gap-junction proteins.     

PACT, a protein activator of the interferon-induced protein kinase, PKR.

PKR, a latent protein kinase, mediates the antiviral actions of interferon. It is also involved in cellular signal transduction, apoptosis, growth regulation and differentiation. Although in virus-infected cells, viral double-stranded (ds) RNA can serve as a PKR activator, cellular activators have remained obscure. Here, we report the cloning of PACT, a cellular protein activator of PKR. PACT heterodimerized with PKR and activated it in vitro in the absence of dsRNA. In mammalian cells, overexpression of PACT caused PKR activation and, in yeast, co-expression of PACT enhanced the anti-growth effect of PKR. Thus, PACT has the hallmarks of a direct activator of PKR.     

GMAP-210, A cis-Golgi network-associated protein, is a minus end microtubule-binding protein

We report that a peripheral Golgi protein with a molecular mass of 210 kD localized at the cis-Golgi network (Rios, R.M., A.M. Tassin, C. Celati, C. Antony, M.C. Boissier, J.C. Homberg, and M. Bornens. 1994. J. Cell Biol. 125:997-1013) is a microtubule-binding protein that associates in situ with a subpopulation of stable microtubules. Interaction of this protein, now called GMAP-210, for Golgi microtubule-associated protein 210, with microtubules in vitro is direct, tight and nucleotide-independent. Biochemical analysis further suggests that GMAP-210 specifically binds to microtubule ends. The full-length cDNA encoding GMAP-210 predicts a protein of 1, 979 amino acids with a very long central coiled-coil domain. Deletion analyses in vitro show that the COOH terminus of GMAP-210 binds to microtubules whereas the NH2 terminus binds to Golgi membranes. Overexpression of GMAP-210-encoding cDNA induced a dramatic enlargement of the Golgi apparatus and perturbations in the microtubule network. These effects did not occur when a mutant lacking the COOH-terminal domain was expressed. When transfected in fusion with the green fluorescent protein, the NH2-terminal domain associated with the cis-Golgi network whereas the COOH-terminal microtubule-binding domain localized at the centrosome. Altogether these data support the view that GMAP-210 serves to link the cis-Golgi network to the minus ends of centrosome-nucleated microtubules. In addition, this interaction appears essential for ensuring the proper morphology and size of the Golgi apparatus.     

Sex hormone-binding globulin, androgen-binding protein, and vitamin K-dependent protein S are homologous to laminin A, merosin, and Drosophila crumbs protein.

Androgen-binding protein (ABP) and sex hormone-binding globulin (SHBG) are extracellular steroid-binding proteins that are homologous to the COOH-terminal domain of vitamin K-dependent protein S, a protein important in blood clotting. We find that the sequences of ABP, SHBG, and protein S are also similar to two basement membrane proteins, laminin and merosin, and to an integral membrane protein, Drosophila crumbs protein. These latter three proteins have important roles in regulating differentiation and development. The sequence similarity corresponds to the G domain of laminin A chain, which binds heparin and type IV collagen. Analysis of a multiple alignment of these proteins reveals one well-conserved segment corresponding to the part of SHBG that binds to its membrane receptor and another corresponding to the part of protein S that binds to C4b-binding protein. The similarities suggest that ABP, SHBG, and protein S may also have functions related to that of laminin and merosin.     

Structure,function, and protein taxonomy

This paper considers two recent arguments that structure should not be regarded as the fundamental individuating property of proteins. By clarifying both what it might mean for certain properties to play a fundamental role in a classification scheme and the extent to which structure plays such a role in protein classification, I argue that both arguments are unsound. Because of its robustness, its importance in laboratory practice, and its explanatory centrality, primary structure should be regarded as the fundamental distinguishing characteristic of protein taxonomy.     

Plastocyanin,an electron-transfer protein

Plastocyanin (Pc) is a copper (Cu)-containing blue protein, that functions as a mobile electron carrier between cytochrome (cyt) f and Photosystem 1 (PS1) in oxygenic organisms. The atomic structure is known and can be described as a -barrel with hydrophobic residues in the interior of the protein. To increase the understanding about structure-function relationships, site-directed mutagenesis of Pc has proven to be very useful. Mainly two spectroscopic techniques, optical and EPR spectroscopy, have been used to investigate how the copper-site is affected by different mutations. The redox properties of the mutants have been investigated and factors that affect the reduction potential are discussed. Absorption and EPR spectra and reduction potentials for the surface mutants are similar to those of the corresponding wild-type. However, mutants around the Cu ion affected the mentioned properties. Comparisons are made with other cupredoxins. Five site-directed mutants of spinach Pc, Pc(Leu12His), Pc(Leu15His), Pc(Thr79His), Pc(Lys81His) and Pc(Tyr83His), have been modified by covalent attachment of a photoactive ruthenium (Ru)-complex at the surface-exposed histidine residues. The rates of the internal electron-transfer reactions exhibit an exponential dependence on the metal-to-metal separation with a decay factor of 1.1 A_-1. A reorganization energy for the Cu-to-Ru electron-transfer reaction of 1.2 eV was determined. Interprotein electron-transfer reactions involving genetically modified Pc are described. Ionic-strength and pH dependencies indicated that electrostatic interactions are involved in the complex formation between Pc and PS 1, which was confirmed by mutations in the acidic patches of Pc. A very specific interaction was further verified by replacements of hydrophobic residues. Position 10, 12, 36, 87 and 90 were found to be very important for the formation of an active complex. A comparison between available structures of Pc and cyt c₆, both effective donors to PS 1, is made. The physiological electron donor to Pc, cyt f, is briefly described.     

Interphotoreceptor retinoid-binding protein. Gene characterization, protein repeat structure, and its evolution

The gene for bovine interphotoreceptor retinoid-binding protein (IRBP) has been cloned, and its nucleotide sequence has been determined. The IRBP gene is about 11.6 kilobase pairs (kb) and contains four exons and three introns. It transcribed into a large mRNA of approximately 6.4 kb and translated into a large protein of 145,000 daltons. To prove the identity of the genomic clone, we determined the protein sequence of several tryptic and cyanogen bromide fragments of purified bovine IRBP protein and localized them in the protein predicted from its nucleotide sequence. There is a 4-fold repeat structure in the protein sequence with 30-40% sequence identity and many conservative substitutions between any two of the four protein repeats. The third and fourth repeats are the most similar pair. All three of the introns in the IRBP gene fall in the fourth protein repeat. Two of the exons, the first and the fourth, are large, 3173 and 2447 bases, respectively. The introns are each about 1.5-2.2 kb long. The human IRBP gene has a sequence that is similar to one of the introns from the bovine gene. The unexpected gene structure and protein repeat structure in the bovine gene lead us to propose a model for the evolution of the IRBP gene.