Dynamic Protein Complexes: Insights from Mass Spectrometry (2001)

mass spectrometry quadrupole time-of-flight The recent sequencing of the human genome has revealed that the human cell contains only twice the number of genes as the worm or fly (1International Human Genome Sequencing Consortium Nature. 2001; 409: 860-921Crossref PubMed Scopus (17495) Google Scholar). This implies that the regulation of gene products as well as their interactions accounts for the increased biological complexity of higher organisms. Consequently to exploit the wealth of information provided by genome sequencing it is essential to be able to study both stable and transient macromolecular complexes. Although the yeast two-hybrid system (2Fields S. Song K.O. Nature. 1989; 340: 245-246Crossref PubMed Scopus (4822) Google Scholar) and cross-linking combined with mass spectrometry (MS)1(3Rappsilber J. Siniossoglou S. Hurt E.C. Mann M. Anal. Chem. 2000; 72: 267-275Crossref PubMed Scopus (172) Google Scholar) are exceptionally powerful approaches to defining stable complexes within the proteasome, MS has the additional potential to describe transient, dynamic complexes through two major developments. These are (i) the ability to probe molecular dynamics through the coupling of MS with hydrogen deuterium (H/D) exchange technologies and (ii) the control of the conditions within the mass spectrometer such that non-covalent interactions between proteins and cofactors can be examined.Over the last decade H/D exchange in conjunction with MS has developed to an extent whereby it can probe the exchange behavior of regions of secondary structure in macromolecular complexes (4Chen J. Smith D. Biochemistry. 2000; 39: 4250-4258Crossref PubMed Scopus (25) Google Scholar) and even individual residues in smaller proteins (5Tito P. Nettleton E.J. Robinson C.V. J. Mol. Biol. 2000; 303: 267-278Crossref PubMed Scopus (39) Google Scholar). In parallel with these developments major advances have been made in the ability to study non-covalent complexes in the gas phase. Specifically the coupling of time-of-flight methods with electrospray and the refinement of this process to a nanoflow technique have enabled applications from simple dimeric complexes (6Vis H. Heinemann U. Dobson C.M. Robinson C.V. J. Am. Chem. Soc. 1998; 120: 6427-6428Crossref Scopus (56) Google Scholar) through to homo- (7van Berkel W.J. van den Heuvel R.H. Versluis C. Heck A.J.R. Protein Sci. 2000; 9: 435-439Crossref PubMed Scopus (115) Google Scholar) and hetero-oligomeric complexes (8Zhang Z. Krutchinsky A. Endicot S. Realini C. Rechsteiner M. Standing K.G. Biochemistry. 1999; 38: 5651-5658Crossref PubMed Scopus (111) Google Scholar) and even small particles (9Tito M.A. Tars K. Valegard K. Hadju J. Robinson C.V. J. Am. Chem. Soc. 2000; 122: 350-351Crossref Scopus (192) Google Scholar, 10Rostom A.A. Fucini P. Benjamin D.R. Juenemann R. Nierhaus K.H. Hartl F.U. Dobson C.M. Robinson C.V. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5185-5190Crossref PubMed Scopus (201) Google Scholar, 11Fuerstenau S.D. Benner W.H. Thomas J.J. Brugidou C. Bothner B. Siuzdak G. Angew. Chem. Int. Ed. Engl. 2001; 40: 542-544Crossref Google Scholar). Consequently the ability to probe both non-covalent interactions as well as H/D exchange enables not only the definition of the stoichiometry of interacting subunits but also their conformational dynamics and subunit interfaces to be investigated.Early applications of mass spectrometry to examine non-covalent protein ligand interactions present in the solution phase led to conflicting results in which specific interactions were not necessarily reproduced in the gas phase of the mass spectrometer. It is now widely accepted that the nature of the interactions, either hydrophobic or ionic, plays a major role in determining the outcome of small protein ligand interactions (12Robinson C.V. Chung E.W. Kragelund B.B. Knudsen J. Aplin R.T. Poulsen F.M. Dobson C.M. J. Am. Chem. Soc. 1996; 118: 8646-8653Crossref Scopus (290) Google Scholar). Specifically, if the dominant interactions are ionic, greater gas phase stability of the complex is observed than for hydrophobically driven ligand binding. With the increasing size of multiprotein complexes generated in the gas phase, however, involving a much greater number of interactions, the net effect is a closer agreement between the gas and solution phase data (13Rostom A.A. Sunde M. Richardson S.J. Schreiber G. Jarvis S. Bateman R. Dobson C.M. Robinson C.V. Proteins Struct. Funct. Genet. 1998; 2 (suppl.): 3-11Crossref Scopus (61) Google Scholar). The fact that these complexes can be observed in the mass spectrometer enables their stability and folding to be probed in the presence of a wide range of ligands and cofactors as well as in response to thermal and chemical denaturation. It is the recent applications of MS to study dynamic protein complexes that form the basis of this review.Binding of Metal Ions and CofactorsDetermining specific binding of metal ions to proteins is now an established application of MS research. In addition to determining the number of metal ions bound specifically to a particular protein the prospect of gaining additional insight into the cooperativity of binding, dissociation constants, and oxidation states of metal ions promises advances in the understanding of protein metal interactions. Using electrospray time-of-flight MS, the stoichiometry of iron binding and subunit association has been confirmed for two hemerythrins and a rubrerythrin (14Lei Q.P. Cui Jr., X. Kurtz D.M. Amster I.J. Chernushevich I.V. Standing K.G. Anal. Chem. 1998; 70: 1838-1846Crossref PubMed Scopus (63) Google Scholar). The major oligomeric complex of hemerythrin from Lingula reevii was observed as a hetero-octamer of α4β4 stoichiometry with one oxygen-bridged diiron site per subunit. By controlling the energy of collisions in the source region, the octamer could be dissociated to a heptamer and monomer from which the metal binding stoichiometry was confirmed as two Fe3+ per monomer. Additionally, there was evidence for binding cooperativity as the intensity of the single bound Fe3+ species was much lower than the apo- or holo-forms. The cooperativity of Zn2+ binding has been demonstrated for human metallothionein (MT-2). From titration of substoichiometric concentrations of Zn2+, cooperative binding of the four-metal cluster was observed to occur first followed by binding of the three-Zn2+ cluster (15Gehrig P.M. You C. Dallinger R. Gruber C. Brouwer M. Kägi J.H.R. Hunziker P.E. Protein Sci. 2000; 9: 395-402Crossref PubMed Scopus (109) Google Scholar). A similar titration approach has been used in a detailed investigation of pH-dependent binding of Cu2+ to synthetic peptides containing the octarepeat sequence from the N-terminal domain of prion protein (16Whittal R.M. Ball H.L. Cohen F.E. Burlingame A.L. Prusiner S.B. Baldwin M.A. Protein Sci. 2000; 9: 332-343Crossref PubMed Scopus (226) Google Scholar). Four octarepeat sections bind two Cu2+ at pH 6.0 and four Cu2+ at pH 7.4. Dissociation constants, calculated from the MS titration data, show that the first two Cu2+ ions are bound more tightly (low micromolar K d) than the third and fourth. However, for the peptide that is extended at the C terminus to include the next seven residues (one additional histidine) the third and fourth Cu2+ are bound cooperatively and have nanomolar dissociation constants (16Whittal R.M. Ball H.L. Cohen F.E. Burlingame A.L. Prusiner S.B. Baldwin M.A. Protein Sci. 2000; 9: 332-343Crossref PubMed Scopus (226) Google Scholar).The specificity of metal-mediated binding of a transcription factor to DNA has been investigated. For the vitamin D receptor DNA-binding domain, binding of the monomer or dimer to its DNA response element was observed only in the presence of Zn2+. However, at higher concentrations of Zn2+ dissociation of the complex occurred. This was attributed to a small conformational change because of occupation of an additional Zn2+-binding site (17Veenstra T.D. Benson L.M. Craig T.A. Tomlinson A.J. Kumar R. Naylor S. Nature Biotech. 1998; 16: 262-266Crossref PubMed Scopus (51) Google Scholar). In an extension to this study the formation of a heterodimer of vitamin D receptor and retinoid X receptor-α in the presence of Zn2+ and the binding of this dimer to the vitamin D response element has been reported (18Craig T.A. Benson L.M. Tomlinson A.J. Veenstra T.D. Naylor S. Kumar R. Nature Biotech. 1999; 17: 1214-1218Crossref PubMed Scopus (35) Google Scholar). Binding of vitamin D to the heterodimeric metalloprotein-DNA complex was also observed, but by contrast addition of retinoic acid was found to disrupt the complex and to favor formation of homodimeric retinoid X receptor-α (18Craig T.A. Benson L.M. Tomlinson A.J. Veenstra T.D. Naylor S. Kumar R. Nature Biotech. 1999; 17: 1214-1218Crossref PubMed Scopus (35) Google Scholar). These results demonstrate the sensitivity of the charge state distribution of both the vitamin D and retinoid X receptors to DNA binding and changes in Zn2+ concentration as well as the specificity of the various components to the presence of retinoic acid.By using a combined MS and limited proteolysis strategy the order of metal binding to multiple sites and the relative stability of these sites can be determined. This approach has been used to study conformational changes on Cu2+ binding in the human copper chaperone, Zn-SOD1 (hCCS); copper is bound by the first and third domain in hCCS, and a cleavage site between the second and third domain is protected from proteolysis when Cu2+ is bound (19Rae T.D. Torres A.S. Pufahl R.A. O'Halloran T.V. J. Biol. Chem. 2001; 276: 5166-5176Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). A comprehensive example of this combined MS/proteolysis method was reported for the 11-kDa estrogen receptor DNA-binding domain (20Whittal R.M. Benz C.C. Scott G. Semyonov J. Burlingame A.L. Baldwin M.A. Biochemistry. 2000; 39: 8406-8417Crossref PubMed Scopus (56) Google Scholar). Oxidation of the estrogen receptor has been implicated in cases where tamoxifen is ineffective in the treatment of breast cancer, and the mass spectrometric study showed that preferential oxidation of one of the two zinc fingers occurred, which prevented dimerization and DNA binding (20Whittal R.M. Benz C.C. Scott G. Semyonov J. Burlingame A.L. Baldwin M.A. Biochemistry. 2000; 39: 8406-8417Crossref PubMed Scopus (56) Google Scholar). Using a similar approach the two zinc fingers in the nucleocapsid protein (NCp7) were examined (21Loo J.A. Int. J. Mass Spectrom. 2001; 204: 113-123Crossref Scopus (37) Google Scholar). The results show that Zn2+ binds preferentially to one zinc finger whereas both are involved in DNA binding as the cleavage site between the Zn2+-binding regions is protected from digestion in a NCp7-pentanucleotide complex (21Loo J.A. Int. J. Mass Spectrom. 2001; 204: 113-123Crossref Scopus (37) Google Scholar).Recent applications have highlighted the ability of MS to determine the oxidation state of metal ions. Using the high resolution capabilities of a Fourier transform mass spectrometer the oxidation state of bound metal ions can be determined by accurate mass measurement of the isotope peaks for a particular charge state. Specifically the heme-containing proteins myoglobin, three different cytochromes (22He F. Hendrickson C.L. Marshall A.G. J. Am. Soc. Mass Spectrom. 2000; 11: 120-126Crossref PubMed Scopus (59) Google Scholar), and rubredoxin (23Johnson K.A. Verhagen M.F.J.M. Brereton P.S. Adams M.W.W. Amster I.J. Anal. Chem. 2000; 72: 1410-1418Crossref PubMed Scopus (61) Google Scholar) have been shown to contain Fe3+. In the latter study, the oxidation state of the iron in a 20-kDa protein containing an iron-sulfur cluster required mass measurement accuracy to within a single mass unit to distinguish proton or metal ligation. A recent report has described the use of an in-line electrochemical cell coupled with electrospray ionization-Fourier transform mass spectrometry that allows observation of metalloproteins in their oxidized or reduced states without the need for anaerobic conditions or chemical reductants (24Johnson K.A. Shira B.A. Anderson J.L. Amster I.J. Anal. Chem. 2001; 73: 803-808Crossref PubMed Scopus (37) Google Scholar). Specifically for the proteins azurin, superoxide reductase, and a mixed iron/manganese-sulfur ferrodoxin, chemical or electrochemical reduction resulted in changes in the oxidation state of the metal ion within the protein complex (24Johnson K.A. Shira B.A. Anderson J.L. Amster I.J. Anal. Chem. 2001; 73: 803-808Crossref PubMed Scopus (37) Google Scholar).Conformational Changes as a Result of PhosphorylationIn addition to monitoring conformational change as a result of metal binding the importance of phosphorylation in signaling pathways has led to the introduction of sensitive methods to monitor conformational change as a result of this modification. Using H/D exchange, limited proteolysis, chromatographic separation of peptides, and an electrospray approach both phosphorylated and non-phosphorylated extracellular signal-regulated protein kinase 2 were examined (25Hoofnagle A. Resing K. Goldsmith E. Ahn N. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 956-961Crossref PubMed Scopus (104) Google Scholar). The results showed that conformational changes observed in the x-ray structures could account for some of the changes observed in H/D exchange rates. For the ATP binding loop, hinge, and substrate binding site, significant differences were observed that could not be explained by the x-ray structure. Significantly these results identify changes in the mobility of the protein in the regions important for catalysis (25Hoofnagle A. Resing K. Goldsmith E. Ahn N. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 956-961Crossref PubMed Scopus (104) Google Scholar). Using a similar approach but analyzing the entire peptide digest in a single matrix-assisted laser desorption ionization spectrum, solvent accessibility was probed in the transient phosphorylated state of the methylesterase CheB (26Hughes C.A. Mandell J.G. Anand G.S. Stock A.M. Komives E.A. J. Mol. Biol. 2001; 307: 967-976Crossref PubMed Scopus (53) Google Scholar). Despite the fact that the lifetime of this state is ∼2 s, comparison with the non-phosphorylated forms reveals that regions in the catalytic domain of this two-domain protein become more solvent-accessible upon phosphorylation (26Hughes C.A. Mandell J.G. Anand G.S. Stock A.M. Komives E.A. J. Mol. Biol. 2001; 307: 967-976Crossref PubMed Scopus (53) Google Scholar). The results of these two studies demonstrate the power of this approach and allow a glimpse of the conformational dynamics of phosphorylated proteins in solution.Protein Misfolding and AggregationThe phenomenon of protein aggregation is a ubiquitous problem in biomedical and biotechnological research. Aggregates can form from the association of transient, partially folded intermediates during refolding experiments and during the production of recombinant proteins in heterologous expression systems (27Wetzel R. Ahern T.J. Manning M.C. Stability of Protein Pharmaceuticals, Part B: In Vivo Pathways of Degradation and Strategies for Protein Stabilization. Plenum Publishing Corp., New York1992: 43-48Google Scholar). Moreover, abnormal protein association is a characteristic of many neurodegenerative conditions including Huntington's, Parkinson's, and Alzheimer's diseases and the transmissible spongiform encephalopathies (28Koo E.H. Lansbury P.T. Kelly J.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9989-9990Crossref PubMed Scopus (589) Google Scholar). For β2-microglobulin, one of a group of 20 or so proteins associated with the formation amyloid fibrils in vivo (29Tan S.Y. Pepys M.B. Histopathology. 1994; 25: 403-414Crossref PubMed Scopus (273) Google Scholar), MS was used to identify N-terminally truncated forms of the protein present in high concentrations in ex vivo deposits (30Bellotti V. Stoppini M. Mangione P. Sunde M. Robinson C.V. Asti L. Brancaccio D. Ferri G. Eur. J. Biochem. 1998; 258: 61-67Crossref PubMed Scopus (102) Google Scholar). The H/D exchange properties of a mixture of wild-type and the major N-terminal truncation of β2-microglobulin showed that the modified protein was significantly less protected from exchange than the wild-type protein (31Esposito G. Michelutti R. Verdone G. Viglino P. Hernández H. Robinson C.V. Amoresano A. Piaz F.D. Monti M. Pucci P. Mangione P. Asti L. Stoppini M. Merlini G. Ferri G. Bellotti V. Protein Sci. 2000; 9: 831-845Crossref PubMed Scopus (218) Google Scholar). Because low protein concentrations in the nanomolar range can be sufficient to prevent aggregation (32Nettleton E.J. Sunde M. Lai V. Kelly J.W. Dobson C.M. Robinson C.V. J. Mol. Biol. 1998; 281: 553-564Crossref PubMed Scopus (153) Google Scholar) MS is well suited for probing the structure of monomeric species. For insulin under amyloidogenic solution conditions (pH 2.0), a total of 19 peptides was generated by peptic digestion and analyzed without chromatographic separation. By subtraction of overlapping peptides the deuterium occupancy of individual residues could be defined and demonstrated that extensive native state helical structure was preserved under these solution conditions (5Tito P. Nettleton E.J. Robinson C.V. J. Mol. Biol. 2000; 303: 267-278Crossref PubMed Scopus (39) Google Scholar). This MS methodology enabled an investigation of the exchange properties of individual residues within a conformation prone to extensive aggregation and ultimately amyloid fibril formation.Further applications of MS to study amyloid formation exploit the ability to monitor non-covalent protein-protein interactions. Spectra of insulin measured directly during the fibril formation process demonstrate the presence of soluble oligomers, up to a 12-mer, their H/D exchange properties being consistent with the reversible association of soluble oligomers (33Nettleton E. Tito P. Sunde M. Bouchard M. Dobson C. Robinson C. Biophys. J. 2000; 79: 1053-1065Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). An MS method for monitoring fibrillogenesis directly within the nanoflow capillary used for introducing the sample into the electrospray source has been demonstrated for the islet amyloid polypeptide (34Larson J.L. Ko E. Miranker A.D. Protein Sci. 2000; 9: 427-431Crossref PubMed Scopus (48) Google Scholar). The results from this investigation showed that the rate of removal of islet amyloid polypeptide in seeded reactions obeys first-order kinetics (34Larson J.L. Ko E. Miranker A.D. Protein Sci. 2000; 9: 427-431Crossref PubMed Scopus (48) Google Scholar). An intriguing series of MS experiments has been designed to probe when a protein is irreversibly committed to either the native or misfolded aggregated state. Briefly, the protocol involves initiation of folding for the 15N-labeled protein under non-aggregating conditions. After various time intervals the experimental conditions are changed rapidly and the protein concentration is increased by the addition of 14N-protein such that monomeric protein begins to aggregate. This allows the proportion of 15N- and 14N-labeled protein, recovered from aggregated and soluble forms, to be determined (35Finke J.M. Gross L.A. Ho H.M. Sept D. Zimm B.H. Jennings P.A. Biochemistry. 2000; 39: 15633-15642Crossref PubMed Scopus (16) Google Scholar). Interestingly for the interleukin 1β variant K97I the results showed that early folding intermediates and aggregates formed in the reaction were reversible but that commitment to aggregation or folding to the native state occurred at a much slower rate (35Finke J.M. Gross L.A. Ho H.M. Sept D. Zimm B.H. Jennings P.A. Biochemistry. 2000; 39: 15633-15642Crossref PubMed Scopus (16) Google Scholar). The of the between protein aggregation and introduction of MS methods for probing both and protein Protein to the of protein folding in vivo is the of a role to molecular The widely of molecular is the and its forms a single of seven subunits The subunits are through to form a structure with a molecular mass of The and of this have been using H/D exchange of in a chemical (4Chen J. Smith D. 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Nature. 1996; PubMed Scopus Google Scholar). By contrast the was found to be but and dissociation of this complex within the cell of a mass spectrometer led to single and monomeric the of the interacting subunits A.A. Robinson C.V. J. Am. Chem. Soc. 1999; Scopus Google protocol of many low energy collisions to the energy of the ions or their dissociation by high energy collisions has been to probe the subunit in a described molecular is an to the involved in the folding of and M. D. K. E. Dobson C.M. Hartl F.U. J. 1999; PubMed Google Scholar). The complex was by first defining the molecular of the complex M. Tito M.A. A.A. Hartl F.U. Dobson C.M. Robinson C.V. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: PubMed Scopus Google Scholar). Although analyzed by size the by the MS method enabled an of the This to a well defined of two subunits and four subunits M. Tito M.A. A.A. Hartl F.U. Dobson C.M. Robinson C.V. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: PubMed Scopus Google Scholar). Dissociation of the complex within the gas phase was used to probe the and two both and four consistent with these were In an extension to this study a nanoflow was to monitor the thermal stability of this The results of these experiments demonstrate that a significant proportion of the under low conditions even at In addition it was to monitor in time the of the from its A mixture of the two subunits in a of subunits was in the nanoflow and the time of the were The mass showed the of that the process is to the In of the x-ray of this complex there are interactions between the subunits the subunits form a dimeric and as a for the of the subunits R. C. Hartl F.U. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). From the MS the cooperative and the stability of the complex at the was as the unit in vivo M. Tito M.A. A.A. Hartl F.U. Dobson C.M. Robinson C.V. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: PubMed Scopus Google the of from its subunits in time by mass the stability of the as a of with from M. Tito M.A. A.A. Hartl F.U. Dobson C.M. Robinson C.V. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: PubMed Scopus Google x-ray structure of the complex R. C. Hartl F.U. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) from the and using the R. M. K. J. Mol. 1996; PubMed Scopus Google when coupled with H/D exchange has been used to examine the of the protein a of the of and a modified protein an additional followed by H/D exchange revealed that exchange of the folded of the protein occurred. By contrast in the presence of and a of ATP the modified protein a protected of that the protein not the results show that with ATP can native proteins to which a peptide has been E. B. Miranker A. A. Nature. 1999; PubMed Scopus Google Protein matrix-assisted laser desorption approach has been developed to probe the protein-protein of protein complexes. that deuterium under H/D exchange conditions because of solvent accessibility at the of the complex were used to report on the interacting regions J. A. Komives E. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). This method has been used to probe complexes of the catalytic subunit of a protein kinase with a kinase The structure of this complex been determined from x-ray data and was used to the MS method the technique to a complex of structure. regions of the protein with solvent accessibility the rapidly of the complex were and with the protocol for the the peptides from the showed a significant in H/D exchange in the complex when with The results from this investigation residues where significant interactions occur between and the of that J. A. Komives E. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google of the MS developments have from the ability to of The of such experiments was first demonstrated by an through the gas phase of a mass spectrometer and that could be G. Bothner B. M. Brugidou C. C.M. K. C.M. Chem. Biol. 1996; Full Text PDF PubMed Scopus Google Scholar). to of particles have the in which individual charge states could be defined in the time-of-flight mass (9Tito M.A. Tars K. Valegard K. Hadju J. Robinson C.V. J.