Amino Acid Sequence Control of Protein Folding, Misfolding and Inclusion Body Formation
The rules through which the linear sequence of amino acids in polypeptide chains determines their three-dimensional fold remain unsolved. The complexity of protein folding processes are clearest when they fail; examples include the accumulation of inclusion bodies instead of folded proteins as the products of cloned genes, and the amyloid family of human diseases.
Triple ß helix as a molecular clamp
The P22 tailspike is one of the few complex proteins whose folding intermediates have been identified both in vivo and in vitro. Each subunit of the trimeric tailspike adhesin includes an elongated processive 13 rung parallel ß helix. Kristen Cook and Cammie Haase-Pettingell are characterizing early intermediates in the in vitro refolding and misfolding of the tailspike chains. After residue 540 the three chains wrap around each other forming an interdigitated triple ß-coil. Formation of this domain proceeds through the protrimer intermediate in the folding pathway, in which the chains are associated but not yet interdigitated. Upon completion of braiding and folding, the melting temperature increases by more than 40ºC, and the mature protein acquires its SDS and protease resistance (Kreisberg, Haase-Pettingell et al, 2002). Thus this motif functions as a molecular clamp, conferring high thermostability on the native trimer. Dr. Peter Weigele has isolated a set of amino acid substitutions within the braided sequence forming the clamp, and shown that a number of these block the transition from the protrimer to the native trimer. The sequence appears to both terminate the single ß-helical fold and direct the strands into the triple-beta-helical motif, conferring the leap in thermostability.
Kinetic role of cysteines in chain folding and assembly
Though the tailspike lacks disulfide bonds, the protrimer intermediate contains interchain disulfide bonds, which must be reduced to form the native trimer. Each of the eight single Cys>Ser mutant proteins was able to fold and assemble at low temperature, and once folded exhibited wild type thermostability and biological activity. Thus the cysteine thiols make only very limited contributions to the stability and activity of the native state. However, the kinetics of the in vitro refolding of the mutant tailspikes were distinctly altered, indicating that these –SH side chains and S-S bonds play critical kinetic roles in productive folding and assembly at physiological temperatures (Haase-Pettingell et al, 2001). We suspect that the transient interchain S-S bonds keep the chains in proper registration in preparation for the wrapping of the chains to form the triple ß-helix. Ryan Simkovsky is investigating the possibility that a thioredoxin motif found in the tailspike sequence is responsible for the disulfide bond chemistry.
Human Gamma-D Crystallins and Lens Cataract Formation
In the human lens cataract formation represents aggregation of the lens crystallins. The 173 amino acid gamma-D crystallin, a two domain all ß-sheet protein with four buried tryptophans, is a major component of cataracts. The human protein has been cloned and expressed by Dr. Ajay Pande, Dr. Jayanti Pande, and Prof. George Benedek in Physics. Inherited juvenile onset cataracts are associated with single amino acid substitutions in human gamma-D crystallin (Pande et al, 2001).
The majority of sight losses from cataracts occur in older adults. Melissa Kosinski-Collins and Shannon Flaugh have established the unfolding and refolding of wild type human gamma-D crystallin using GdnHCl as the denaturing agent. A distinctive aggregation/polymerization pathway of a non-native conformer competes with refolding, and may provide insight into cataractogenesis. The unfolded protein is more fluorescent than its native counterpart despite the apparent lack of any obvious metal or cofactor interactions. Construction of four triple phenylalanine substitutions leaving a single tryptophan has been completed. Characterization of these single tryp proteins reveals that two of the tryptophans are responsible for the anomalous fluorescence behavior. The role of single tryptophans in crystallin stability has been investigated by Veronica Zepeda, who has constructed and characterized single Tryp>Ala mutants.
Shannon Flaugh has constructed and characterized mutants of amino acid residues at the domain interface of the crystallin. In some models of cataractogenesis, such as domain swapping, this interface would play a key role in the precursor to the cataract state. Her interface mutants fold into the native state but exhibit subtle alterations in their equilibrium unfolding and refolding. Shannon and Veronica have also shown that the human gamma-D crystallin protein is very thermostable, with a melting temperature of above 75°C. The contributions of various buried side chains to thermal stability is under investigation.
Prof. Bonnie Berger in Applied Mathematics together with Prof. Lenore Cowan (Tufts), Matt Menke, and Phil Bradley (now at UW Seattle) have led a collaborative effort to develop algorithms that can identify sequences which will fold into parallel ß-coils. The program, Betawrap, successfully finds the known ß-helices in the Protein Data Bank with no false negatives or positives. When run on genebank sequences, it scores very highly a few hundred sequences almost all of which are from microbial species, including numerous human pathogens. This may reflect the special function of parallel ß coils in binding long floppy polysaccharides important in host cell recognition.
In collaboration with Judith Klein-Seetharaman and Deborah Weisser at Carnegie-Mellon and University of Pittsburgh Medical Center, Dr. Peter Weigele and Welkin Pope are using biological language modeling tools to examine the sequence control of ß-helix and triple ß-helix formation.
In collaboration with Prof. David Gossard in Mechanical Engineering, we are developing kinematic models for subunit polymerization into icosahedral shells, as well as the lattice transformations required for the procapsid to capsid transition in dsDNA viruses. These lattice transformations provide a model for a very general set of protein subunit conformational changes that occur not in solution but in constrained polymeric structures.
Protein folding in Ecologically Important Marine Cyanobacteria and their phages
Dr. Claire Ting, in collaboration with Prof. Penny Chisholm, has studied the photosynthetic apparatus and heat shock response of the important marine bacteria Prochlorococcus and Synechococcus. Welkin Pope and Dr. Peter Weigele are characterizing phages of marine Synechococcus to examine the sequence/structure relationships of their adhesins.
Pope, WH, Weigele, PR, Chang, J, Pedulla, ML, Ford, ME, Houtz, JM, Jiang, W, Chiu, W, Hatfull, GF, Hendrix, RW and King, JA (2007), Genome sequence, structural proteins, and capsid organization of the cyanophage Syn5: a "horned" bacteriophage of marine Synechococcus. J. Mol. Biol., 368, 966-981.
McDonnell, A.V., Menke, M., Palmer, N., King, J., Cowen, L. and Berger, B. (2006) Fold recognition and accurate sequence-structure alignment of sequences directing b-sheet proteins. Proteins, 63, 976-985.
Flaugh, S.L., Mills, I.A. and King, J. (2006) Glutamine deamidation destabilizes human gD-crystallin and lowers the kinetic barrier to unfolding. JBC, 281, 30782-30793.
Chen, J., Flaugh, S.L., Callis, P.R. and King, J. (2006) Mechanism of the highly efficient quenching of tryptophan fluorescence in human gD-crystallin. Biochemistry, 45, 11552-11563.
Chang, J., Weigele, P., King, J., Chiu, W. and Jiang, W. (2006) Cryo-EM asymmetric reconstruction of bacteriophage P22 reveals organization of its DNA packaging and infecting machinery. Structure, 14, 1073-1082.
Simkovsky, R. and King, J. (2006) An elongated spine of buried core residues necessary for in vivo folding of the parallel b-helix of P22 tailspike adhesin. PNAS, 103, 3575-3580.
Schwartz, R. and King, J. (2006) Frequencies of hydrophobic and hydrophilic runs and alternations in proteins of known structure. Protein Science, 15, 102-112.
Jiang, W., Chang J., Jakana, J., Weigele, P., King, J. and Chiu, W. (2006) Structure of complete Epsilon 15 phage reveals organization of condensed DNA and DNA packaging/injection apparatus. Nature, 439, 612-616
Weigele, P., Haase-Pettingell, C., Campbell, P.G., Gossard, D.C. and King, J. (2005) Stalled folding mutants in the triple b-helix domain of the phage P22 tailspike adhesin. J.Mol.Biol., 354: 1103-1117.
Flaugh, S., Kosinski-Collins, M. and King, J. (2005) Inter-domain side chain interactions in human gD-crystallin influencing folding and stability. Protein Science, 14: 2030-2043.
Jain, M., Evans, M.S., King, J. and Clark, P.L. (2005) Monoclonal epitope mapping describes tailspike -helix folding and aggregation intermediates. J.Biol.Chem., 280: 23032-23040.
Flaugh, S., Kosinski-Collins, M and King, J. (2005) Contributions of hydrophobic domain interface interactions to the folding and stability of human gammaD-crystallin. Protein Science, 14: 569-581.
Kosinski-Collins, M, Flaugh, S. and King, J. Probing folding and fluorescence quenching in human gammaD-crystallin Greek key domains using triple tryptophan mutant proteins. Protein Science, 13: 2223-2235.
Betts, S., Haase-Pettingell, C., Cook, K. and King, J. (2004) Buried hydrophobic side chains essential for the folding of the parallel b-helix domains of the P22 tailspike. Protein Science, 13: 2291-2303.
Kosinski-Collins, M. & King, J. (2003) In vitro unfolding and refolding of human gamma-D crystallin, a protein involved in cataract formation. Protein Science, 12: 480-490.
Cowen, L., Bradley, P., Menke, M., King, J. and Berger, B. (2002) Predicting the beta-helix fold from protein sequence data. J. Comput. Biol., 9: 261-276.
King, J., Haase-Pettingell, C. & Gossard, D. (2002) Protein Folding and Misfolding. American Scientist, 90: 445-453.
Kreisburg, J.F., Betts, S. D., Haase-Pettingell, C. and King, J. (2002) The interdigitated beta-helix domain of the P22 tailspike protein acts as a molecular clamp in trimer stabilization. Protein Science, 11: 820-830.
Bradley, P., Cowen, L., Menke, M., King, J. and Berger, B. (2001) BetaWrap: Successful prediction of parallel ß-helices from primary sequence reveals an association with many microbial pathogens. Proc. Natl. Acad. Sci. USA, 98: 14819-14824.
Clark, P.L. and King, J. (2001) A newly synthesized, Ribosome-bound polypeptide chain adopts conformations dissimilar from early in vitro refolding intermediates. J. Biol.Chem., 276: 25411-25420.
Pande, A., Pande, J., Asherie, N., Lomakin, A., Ogun, O., King, J. and Benedek, G. (2001) Crystal cataracts: Human genetic cataract due to protein crystallization. Proc. Natl. Acad. Sci. USA, 98: 6116-6120.
Raso, S.W., Clark, P.L., Haase-Pettingell, C., King, J. and Thomas, G.J., Jr. (2001) Distinct cysteine sulfhydryl environments detected by analysis of raman S-H markers of Cys —> Ser mutant proteins. J. Mol. Biol., 307: 899-911.
Haase-Pettingell, C., Betts, S., Raso, S.W., Stuart, L., Robinson, A. and King, J. (2001) Role for cysteine residues in the in vivo folding and assembly of the phage P22 tailspike. Protein Science, 10: 397-410.
Raso, S.W. and King, J. (2000) Protein folding and human disease. In: Frontiers in Molecular Biology: Mechanisms of Protein Folding, 2nd Edition (ed. R.H. Pain), Oxford University Press, pp 406-428.