Subunit structure of human erythrocyte glycophorin A.

Glycophorin A is a sialoglycoprotein isolated from human erythrocyte membranes which seems to exist as stable dimeric complexes in the presence of sodium dodecyl sulfate. When analyzed by dodecyl sulfate acrylamide electrophoresis this molecule forms two PAS-stainable bands (PAS-U and PAS-2) which are reversibly interconvertible. This change in electrophoretic mobility is dependent on the concentration of dodecyl sulfate, the use of Trisbuffer systems, the protein concentration in the incubation mixture, and the duration and temperature of incubation before electrophoresis. Reducing agents do no influence the results. Chromatography of the sialoglycopeptides on Sepharose columns in dodecyl sulfate before and after heat treatment gave similar results. A small hydrophobic peptide (T-6) derived from glycophorin A was able to prevent reassociation of the monomeric subunits back to the higher molecular weight form. This peptide was able to bind to the subunit of glycophorin A, but not to the high molecular weight complex. These results are consistent with a model of glycophorin A composed of two subunits which can dissociate and reassociate in the presence of detergents. These subunits may interact via the hydrophobic portions of the polypeptide chains.     

1H-Nuclear magnetic-resonance studies on glycophorin and its carbohydrate-containing tryptic peptides.

The proton nuclear magnetic resonance (1H-NMR) spectra of glycophorin and its tryptic sialoglycopeptides were investigated. From the intensities of the assigned resonances it was concluded that all of the residues in the sialoglycopeptides are sufficiently mobile in conformation to give sharp resonances, while in glycophorin this is true for only approximately 80% of the peptide backbone. The resonances of the central sequence of some 20 of the hydrophobic residues are strongly broadened. This region is probably that of alpha-helical structure which is known to aggregate. The linewidths and intensities of the resonances are not, or only slightly, affected by changing the ionic strength, temperature or by carboxymethylation of the Met-81 residue in glycophorin. Glycophorin was found to bind about 100 mol sodium dodecylsulphate/mol protein as derived from studies on linebroadening of the latter's C-3 to C-11 methylene resonances. The bound dodecyl-sulphate probably increases the mobilities of the hydrophobic residues in the protein as these resonance intensities are increased by the binding. The carbohydrate chains in glycophorin were conformationally mobile; no evidence was found for tight carbohydrate-protein interactions. The relevance of flexible carbohydrate chains in membrane glycoproteins is discussed in relation to cell surface chemistry.     

Enzymatic transfer of ATP gamma S thiophosphate onto the 5'-hydroxyl of an oligonucleotide as a route to reactive oligonucleotide derivatives

Carbon-13 nuclear magnetic resonance spectroscopy was used to monitor the preferential sulfoxidation of the two methionine residues (8 and 81) of glycophorin A. In urea Met-8 is readily oxidized. However, Met-81 can only be oxidized in trifluoroacetic acid containing hydrogen peroxide. Our results also give some insight into the reagent accessibility of different portions of the protein molecule and the general stability of this glycoprotein.     

Characterization of the dimerization domain in the FNR transcription factor

The global anaerobic regulator FNR from Escherichia coli is a dimeric Fe-S protein that is inactivated by O(2) through disruption of its [4Fe-4S] cluster and conversion to a monomeric form. As a first step in elucidating the molecular interactions that control FNR dimerization, we have performed alanine-scanning mutagenesis of a potential dimerization domain. Replacement of many hydrophobic residues (Met-143, Met-144, Leu-146, Met-147, Ile-151, Met-157, and Ile-158) and two charged residues (Arg-140 and Arg-145) with Ala decreased FNR activity in vivo. Size exclusion chromatography and Fe-S cluster analysis of three representative mutant proteins, FNR-M147A, FNR-I151A, and FNR-I158A, showed that the Ala substitutions produced specific defects in dimerization. Because hydrophobic side chains are known to stabilize subunit-subunit interactions between alpha-helices, we propose that Met-147, Ile-151, and Ile-158 lie on the same face of an alpha-helix that constitutes a dimerization interface. This alignment would also position Arg-140, Met-144, and Asp-154 on the same helical face. In support of the unusual positioning of a negatively charged residue at the dimer interface, we found that replacing Asp-154 with Ala repaired the defects caused by Ala substitutions of other residues located on the same helical face. These data also suggest that Asp-154 has an inhibitory effect on dimerization, which may be a key element in the control of FNR dimerization by O(2) availability.     

Protein chromatography on adsorbents with hydrophobic and ionic groups. Purification of human erythrocyte glycophorin.

Human erythrocyte glycophorin was purified rapidly by (a) chromatography of a Triton X-100 extract of erythrocyte 'ghosts' on N-(3-carboxypropionyl)aminodecyl-Sepharose in buffers containing Triton X-100 or sodium dodecyl sulphate, or (b) chromatography of whole 'ghosts', solubilized in sodium dodecyl sulphate, on dodecyl-Sepharose, in buffers containing sodium dodecyl sulphate. The products contained 85-95% glycophorin (electrophoretic band PAS-1) and the major contaminants were glycoproteins PAS-2 (possibly a subunit of glycophorin) and PAS-3.     

A proton nuclear magnetic resonance and molecular modeling study of calmidazolium (R24571) binding to calmodulin and skeletal muscle troponin C

1H NMR spectroscopy at 360 MHz has been used to study the interactions between the calmodulin function inhibitor calmidazolium (R24571) and (i) calmodulin (CaM) and (ii) skeletal muscle troponin C (sTnC). One equivalent of racemic calmidazolium binds tightly to CaM and perturbs a number of protein signals, corresponding to residues in both dicalcium-binding domains, in a manner characteristic of slow exchange. Calmidazolium binds with lower affinity to sTnC but still induces widespread perturbations in both domains. Extensive spectral overlap precludes definite assignment of intermolecular nuclear Overhauser effect (NOEs) although intraprotein NOEs do indicate the nature of some drug-induced conformational changes. Relaxation enhancements induced by two spin-labeled calmidazolium analogues demonstrate that several methionine residues of CaM, significantly immobilized by calmidazolium binding, are in fact located at or near its binding sites. These and other residue-specific broadening effects have enabled low resolution models to be constructed of the predominantly hydrophobic drug-binding sites on each domain of CaM. The hydrophobic portions of calmidazolium itself, and its analogues, contact side chains of Ala-15, Leu-18, Phe-19, Val-35, Met-36, Leu-37, Leu-39, Met-51, Met-71, Met-72, and Met-76 in the N-terminal domain of calmodulin, and Ala-88, Val-91, Phe-92, Val-108, Met-109, Leu-112, Phe-141, and Met-145 in its C-terminal domain. The model, and an analogous one of sTnC, can be used to rationalize drug-induced changes in intraprotein NOEs. Issues pertaining to the possible simultaneous binding of calmidazolium to both globular domains of the proteins are discussed in terms of the experimental results and the overall structures of each protein.     

Operation of the glucose-fatty acid cycle after glycogenolysis induced by treatment with nicotinic acid.

The intramembranous segment of glycophorin A has been localized to a 35-amino acid peptide. This has been isolated by a new procedure in which acid-insoluble peptides of a tryptic digest of detergent-purified glycophorin A are fractionated by countercurrent distribution. Amino acid sequence analyses, using both manual and automatic Edman degradation techniques, indicate that this peptide has a unique sequence in contrast to earlier work (J. P. Segrest, I. Kahane, R. L. Jackson, and V. T. Marchesi, 1973, Biochem. Biophys. Res. Commun., 49, 964–969). Ambiguities at three positions have been resolved, and sequencing errors at two additional positions have been corrected. One segment of this peptide has an uninterrupted stretch of 22 uncharged amino acids, and it is likely that this is the part which spans the lipid bilayer of the membrane. The complete 35-residue peptide has an apparent molecular weight in the 6000–8000 range, when analyzed on sodium dodecyl sulfate gels, suggesting that it forms dimers under these conditions. This result is consistent with our earlier proposal that intact glycophorin A molecules exist as dimers in sodium dodecyl sulfate which are stabilized by noncovalent associations between hydrophobic segments of their polypeptide chains.     

The use of UV-visible spectroscopy for the determination of hydrophobic interactions between neuropeptides and membrane model systems.

Ultraviolet-visible spectroscopy has been used as a rapid method to evaluate the hydrophobic interactions between a series of cationic and zwitterionic neuropeptides and dipeptides with the hydrophobic core of two membrane model systems; sodium dodecyl sulfate and lysophosphatidylcholine micelles. If a hydrophobic interaction occurs, a 1-nm bathochromic shift is observed in the uv-visible spectrum of the aromatic side chains when going from aqueous solution to a micellar solution. The aromatic residues of substance P, bradykinin, and Des-Arg9 bradykinin all exhibited the 1-nm bathochromic shift in the presence of sodium dodecyl sulfate while those of Met-enkephalin did not. The opposite effects were observed in the presence of lysophosphatidylcholine micelles.     

Predicted secondary and tertiary structures of carp gamma-crystallins with high methionine content: role of methionine residues in the protein stability.

A systematic structural comparison of several carp gamma-crystallins with high methionine contents was made by the secondary-structure prediction together with computer model-building based on the established X-ray structure of calf gamma-II crystallin. The overall surface hydrophilicity profile and the distribution of helices, beta-sheets, and beta-turns along the polypeptide chains are very similar among these carp gamma-crystallins. In addition, their general polypeptide packing is close to the characteristic 2 domain/4 motif Greek key three-dimensional conformation depicted for the calf gamma-II crystallin. Interestingly, most hydrophobic methionine residues are located on the protein surface with only a few buried inside the protein surface or in the interface between two motifs of each domain. The exposed hydrophobic and polarizable methionine cluster on the protein surface may have a bearing on the crystallin stability and dense packing in the piscine species, and probably also provides a malleable nonpolar surface for the interaction with other crystallin components for the maintenance of a clear and transparent lens.     

The hemoglobins of phoronopsis viridis, of the primitive invertebrate phylum phoronida: characterization and subunit structure.

The primitive invertebrate, Phoronopsis viridis, of the phylum Phoronida, has intra-cellular hemoglobins composed of four unique polypeptide chains, two of which associate to form hetero- and homodimers and two which do not associate at all. The CO-derivatives of the associating chains are completely dimeric; removal of the ligand does not result in further aggregation as it does in several other invertebrate hemoglobins. Oxidation of the associating hemoglobins is accompanied by dissociation to monomers, but the cyanide derivative of the methemoglobin is dimeric. The four polypeptide chains all have molecular weights of about 16,000 as determined by iron content and gel electrophoresis with sodium dodecyl sulfate. The two associating chains form three components with isoelectric points at pH 5.6, 5.9, and 6.9 whereas those for the two monomeric chains are at pH 6.2 and 7.9. The chains have been characterized by amino acid composition, tryptic peptide patterns, and the amino acid sequence of the NH₂-terminal segment. The oxygen equilibrium of a dimeric fraction has been determined at pH 7.5 and 20 °C; the pressure of half-saturation is 2.3 mm Hg.     

Chemistry and subunit structure of yeast hexokinase isoenzymes

Evidence from ultracentrifugation, sodium dodecyl sulfate electrophoresis, peptide mapping, and carboxypeptidase A digestion allows the conclusion that the two native hexokinases, P-I and P-II, consist of polypeptide chains having molecular weights slightly higher than 50,000. It was demonstrated that some preparations are contaminated with a protease, and that this impurity caused erroneous results in sodium dodecyl sulfate electrophoresis and carboxypeptidase A digestion.Amino acid analyses indicated that both P-I and P-II contain four cysteine, four tryptophan, and eleven methionine residues per mole. In contrast, P-I contains eight, and P-II five, histidine residues per mole. Many of the differences in amino acid composition are small, but reproducible.Peptide mapping indicated that many segments of P-I and P-II have identical sequences. There were about 27 common tryptic peptides, and about 16–19 unique to each form. In addition, both isozymes were found to have the same amino terminus, valine, and the same carboxy terminus, alanine; some evidence for a difference in the penultimate residue at the carboxy terminus was indicated.     

Detergents modulate dimerization, but not helicity, of the glycophorin A transmembrane domain.

Understanding how the lipid environment influences transmembrane helix association requires thermodynamic measurements that can be interpreted in terms of specific chemical interactions. We have used F?rster resonance energy transfer to measure dimerization of the glycophorin A transmembrane helix in detergent micelles. The observed Kd is at least two orders of magnitude weaker in sodium dodecyl sulfate than it is in zwitterionic detergents. In contrast, neither dimerization nor the detergent affects the secondary structure of the glycophorin A helix as measured by far-UV circular dichroism. These measurements support a long standing assumption about the glycophorin A transmembrane domain, that detergents uncouple helix formation from helix dimerization. The approach is applicable to a variety of systems in diverse environments, extending our ability to measure how interactions with complex solvents affect the thermodynamics of oligomerization.     

Retention of native-like oligomerization states in transmembrane segment peptides: application to the Escherichia coli aspartate receptor

Biophysical study of the transmembrane (TM) domains of integral membrane proteins has traditionally been impeded by their hydrophobic nature. As a result, an understanding of the details of protein-protein interactions within membranes is often lacking. We have demonstrated previously that model TM segments with flanking cationic residues spontaneously fold into alpha-helices upon insertion into membrane-mimetic environments. Here, we extend these studies to investigate whether such constructs consisting of TM helices from biological systems retain their native secondary structures and oligomeric states. Single-spanning TM domains from the epidermal growth factor receptor (EGFR), glycophorin A (GPA), and the influenza A virus M2 ion channel (M2) were designed and synthesized with three to four lysine residues at both N- and C-termini. Each construct was shown to adopt an alpha-helical conformation upon insertion into sodium dodecyl sulfate micelles. Furthermore, micelle-inserted TM segments associated on SDS-PAGE gels according to their respective native-like oligomeric states: EGFR was monomeric, GPA was dimeric, and M2 was tetrameric. This approach was then used to investigate whether one or both of the TM segments (Tar-1 and Tar-2) from the Escherichia coli aspartate receptor were responsible for its homodimeric nature. Our results showed that Tar-1 formed SDS-resistant homodimers, while Tar-2 was monomeric. Furthermore, no heterooligomerization between Tar-1 and Tar-2 was detected, implicating the Tar-1 helix as the oligomeric determinant for the Tar protein. The overall results indicate that this approach can be used to elucidate the details of TM domain folding for both single-spanning and multispanning membrane proteins.     

Human adenine phosphoribosyltransferase. Affinity purification, subunit structure, amino acid composition, and peptide mapping.

Adenine phosphoribosyltransferase (EC 2.4.2.7) has been purified 55,000-fold from normal human erythrocytes. The native molecular weight of the enzyme is 38,200 as determined by sedimentation equilibrium centrifugation. The subunit molecular weight is 18,000 as determined by sodium dodecyl sulfate gel electrophoresis and 17,000 as determined by gel filtration in guanidine hydrochloride, suggesting that the enzyme is a dimer in its native state. Cross-linking the enzyme with dimethylsuberimidate confirms the dimeric structure and peptide mapping data suggested that the subunits are quite similar if not identical. The amino acid composition reveals that 33% of the residues are hydrophobic.     

Myosin light chain kinase binding to plastic

Methionine-81 and/or -8 of the transmembrane sialoglycoprotein, glycophorin A, have been specifically alkylated with ¹³CH₃I to produce the sulfonium ion derivatives [S-[¹³C]methylmethionine-8]glycophorin A and [S-[¹³C]methylmethionine-8 and -81]glycophorin A. ¹³C NMR spectra of these species show that the resonances of the methyl groups of the modified glycophorins occur at 26.1 ppm downfield from Me₄Si. A spin-lattice relaxation time of 0.4 was observed for the ¹³C-enriched methyl resonances of the sulfonium ion derivatives of Met-8 and -81, which corresponds to an effective correlation time of < 2× 10^?10 s. Demethylation of the 2 glycophorin A sulfonium ion species with 2-mercaptoethanol produces native glycophorin A which now has the ε-carbon of the methionine residue(s) 45% isotopically enriched. The ε-carbon of Met-8 was found to occur at 15.7 ppm downfield from Me₄Si whereas the ε-carbon of Met-81 exhibited an unusual chemical shift of 2.0 ppm downfield from Me₄Si. The spin-lattice relaxation time of both resonances was found to be ～0.3 s.     

Isolation of the major intrinsic transmembrane protein of the human erythrocyte membrane.

The major intrinsic protein of the human erythrocyte membrane commonly referred to as "Band 3", was isolated by a multi-step procedure. Extraction of ghost membranes in dilute solutions of lithium diiodosalicylate removed most of the proteins considered to be extrinsic to the membrane. The resulting membrane fragments were solubilized in sodium dodecyl sulfate, and the major sialoglycoprotein (glycophorin A) was removed by wheat germ agglutinin-Sepharose affinity chromatography. Gel filtration in sodium dodecyl sulfate was used as the final step to yield the band 3 polypeptide in electrophoretically homogeneous form.     

The interdigitated beta-helix domain of the P22 tailspike protein acts as a molecular clamp in trimer stabilization

The P22 tailspike adhesin is an elongated thermostable trimer resistant to protease digestion and to denaturation in sodium dodecyl sulfate. Monomeric, dimeric, and protrimeric folding and assembly intermediates lack this stability and are thermolabile. In the native trimer, three right-handed parallel beta-helices (residues 143-540), pack side-by-side around the three-fold axis. After residue 540, these single chain beta-helices terminate and residues 541-567 of the three polypeptide chains wrap around each other to form a three-stranded interdigitated beta-helix. Three mutants located in this region -- G546D, R563Q, and A575T -- blocked formation of native tailspike trimers, and accumulated soluble forms of the mutant polypeptide chains within cells. The substitutions R563Q and A575T appeared to prevent stable association of partially folded monomers. G546D, in the interdigitated region of the chain, blocked tailspike folding at the transition from the partially-folded protrimer to the native trimer. The protrimer-like species accumulating in the G546D mutant melted out at 42 degrees C and was trypsin and SDS sensitive. The G546D defect was not corrected by introduction of global suppressor mutations, which correct kinetic defects in beta-helix folding. The simplest interpretation of these results is that the very high thermostability (T(m) = 88 degrees C), protease and detergent resistance of the native tailspike acquired in the protrimer-to-trimer transition, depends on the formation of the three-stranded interdigitated region. This interdigitated beta-helix appears to function as a molecular clamp insuring thermostable subunit association in the native trimer.     

The key role of the residue 103 in the surface interactions of gamma-crystallins

A comparative study of intramolecular crystal interactions of two homologous gamma-crystallins II and IIIb from calf lens has been carried out. It has been shown that the key role in formation of "dimeric" associates of the head-to-tail type for gamma-crystallin IIIb is played by Met-103 which is located in the middle of the hydrophobic surface region. The absence of such a region in the molecule of gamma-crystallin II is explained by replacement of Met-103 by Ser-103. A similar alternative with the exchange of the hydrophobic residue by the hydrophilic one is observed for different gene products of gamma-crystallins from a number of vertebrates. This suggests intermolecular interaction of gamma-crystallins in the native medium of the lens.     

Polar residue tagging of transmembrane peptides

Studies that focus on packing interactions between transmembrane (TM) helices in membrane proteins would greatly benefit from the ability to investigate their association and packing interactions in multi-spanning TM domains. However, the production, purification, and characterization of such units have been impeded by their high intrinsic hydrophobicity. We describe the polar tagging approach to biophysical analysis of TM segment peptides, where incorporation of polar residues of suitable type and number at one or both peptide N- and C-termini can serve to counterbalance the apolar nature of a native TM segment, and render it aqueous-soluble. Using the native TM sequences of the human erythrocyte protein glycophorin A (GpA) and bacteriophage M13 major coat protein (MCP), properties of tags such as Lys, His, Asp, sarcosine, and Pro-Gly are evaluated, and general procedures for tagging a given TM segment are presented. Gel-shift assays on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) establish that various tagged GpA TM segments spontaneously insert into micellar membranes, and exhibit native TM dimeric states. Sedimentation equilibrium analytical centrifugation is used to confirm that Lys-tagged GpA peptides retain the native dimer state. Two-dimensional nuclear magnetic resonance (NMR) spectroscopy studies on Lys-tagged TM MCP peptides selectively enriched with N-15 illustrate the usefulness of this system for evaluating monomer-dimer equilibria in micelle environments. The overall results suggest that polar-tagging of hydrophobic (TM) peptides approach constitutes a valuable tool for the study of protein-protein interactions in membranes.     

Subunit structure of Mycobacterium smegmatis fatty acid synthetase. Evidence for identical multifunctional polypeptide chains

Fatty acid synthetase from Mycobacterium smegmatis has been purified to near homogeneity as judged by a variety of electrophoretic criteria under both native and dissociating conditions. A single protein band was obtained on gel electrophoresis in sodium dodecyl sulfate or 8 M urea at various pH values and on isoelectric focusing in 8 M urea. A subunit molecular weight of about 290,000 was found by polyacrylamide gel electrophoresis in sodium dodecyl sulfate or by sedimentation equilibrium ultracentrifugation in 6 M guanidine HCl. Quantitative Quantitative determination of pantetheine, of flavin, and of the number of fatty acids synthesized during a single enzyme turnover all yield values corresponding to a stoichiometry of about 1 mol per mol of subunit, providing strong evidence that M. smegmatis fatty acid synthetase is an oligomer of identical, multifunctional polypeptide chains.