Biophysical Studies of Protein Folding, Aggregation and Interactions with Molecular Chaperones

Abstract: The information for a protein to fold correctly into a well defined three-dimensional functional structure is embedded in the primary sequence. Much effort has been devoted to characterize the nature of the unfolded state, since this state represents the starting point of the folding reaction. Also the formation of protein aggregates has an important role in biotechnology and also causes numerous diseases. Nature has evolved molecular chaperones as a protection system to prevent protein aggregation. By studying the interactions between chaperones and their protein substrates many important clues about protein folding and misfolding can be found.It is very difficult to structurally investigate partially folded/unfolded proteins, because of their high intrinsic dynamics. Therefore, to obtain site specific information of the protein structure a cysteine labeling approach together with spectroscopic methods can be used. By this approach cysteines can be introduced at particular locations in the protein structure. The sulfhydryl groups of these Cys residues are either used as handles to which various spectroscopic labels can be attached or the chemical reactivity of the sulfhydryl group by itself constitute the probe function. Spin and fluorescent labels will provide dynamic information and report on changes in mobility and polarity. Communicating labels, such as pyrene-pyrene excimer fluorescence and fluorescence resonance energy transfer, FRET, can be used to monitor distances within proteins. In this thesis human carbonic anhydrase II, HCA II, has been used as a model protein for studies of protein folding, aggregation and interactions with the molecular chaperone GroEL.Residual structure in the unfolded state of HCA II: HCA II consists of 259 amino acids and folds into a mainly β-sheet protein. Ten β-strands span the central part of the protein and divide the molecule into two halves, one half containing the active site and the N-terminal subdomain, and the other half containing a large hydrophobic core. By utilization of a doubly labeled cysteine mutant (N67C/C206) labeled with pyrene fluorophores the unfolding process of the central part of HCA II could be monitored by the loss of pyrene excimer fluorescence separating the pyrenes. These measurements revealed an unfolding transition at a GuHC1 concentration significantly higher than that required to induce unfolding of the protein as monitored by circular dicrosim, CD. Utilizing fluorescence resonance energy transfer, FRET, between tryptophans and an inserted acceptor (AEDANS) at various positions (16, 54, 67, 79, 118, 142,146,244 and 245) in the protein revealed a continuous solvation of the central core of HCA II at very strong GuHC1 concentrations. In addition, the use of a single site (position 79) labeled with different probes in the periphery of the central hydrophobic core showed that a persistent cluster can form locally and be associated to the central core. These results provide a picture of stable structures within HCA II mostly composed of hydrophobic clusters that suggestively guide the folding process at the onset of folding.Aggregation of HCA II: Partial unfolding of HCA II results in a molten-globule intermediate, believed to retain native-like secondary structure but very little tertiary interactions. This molten-globule intermediate of HCA II was found to form aggregates. Local and long-range interactions in a protein sequence form a pattern of favorable interactions strongly dependent on each other, meaning that the protein folding process is highly cooperative. These intramolecular interactions are a prerequisite for correct folding. However, if such high affinity exists within a single protein chain, another protein molecule with the same exposed structural pattern can instead form intermolecular interactions with the first protein chain. This will result in the formation of protein oligomers and aggregates. During denaturation by 1-2 M GuHC1 HCA II forms aggregates that organize into an ensemble of soluble oligomers. During thermal denaturation HCA II forms aggregates that grow into micron sized precipitates. The aggregates are composed of partially folded proteins with a distribution of polar, dynamic as well as non-polar, compact regions. The intermolecular interactions involved in aggregate formation of HCA II were localized in a direct way by measuring pyreneexcimer formation between each of 20 site-specific pyrene-labeled cysteine mutants. Another approach was the utilization of 9 site-specific AEDANS-labeled cysteine mutants for tryptophan-AEDANS FRET measurements. The data showed that the contact area of the aggregated protein was very specific, and all sites included in the intermolecular interactions were located in the large β-sheet of the protein, within a limited region between the central β-strands 4 and 7. This substructure is very hydrophobic, which underlines the importance of hydrophobic interactions between specific β-sheet containing regions inaggregate formation.GroEL-HCA II interactions: Aggregation of HCA II is prevented by the presence of the chaperon in GroEL,and because GroEL and a molten-globule intermediate of HCA II form a complex at elevated temperatures the interactions between the two proteins can be studied. These interactions were mapped by site-directed spin-labeling and EPR (Electron Paramagnetic Resonance) measurements and by site-directed fluorescencelabeling and fluorescence shift and AEDANS FRET measurements. The interaction with GroEL was shown to include interactions with outer parts of the HCA II molecule, such as peripheral β-strands and the N-terminal domain, which have previously been shown to be rather unstable. As a result of the interaction, the rigid and compact hydrophobic core was shown to exhibit higher flexibility. FRET measurements showed that the volume of HCA II expanded significantly compared to the molten-globule intermediate as a result of the interaction with GroEL. This suggests an active role of GroEL when exerting its chaperone action and the unfolding action is likely to facilitate rearrangements of misfolded structure in the substrate protein. How does GroEL exert this unfoldase activity? By the use of site-specific labeling of cysteines in GroEL it was possible to observe conformational changes induced by HCA II binding. i) Using iodoacetate to measure cysteine reactivity showed that the cysteines became more accesible during HCA II binding. ii)Determining reactivity with spin labels and thereafter performing EPR measurements demonstrated that the cysteines in GroEL were buried and became accessible upon binding of HCA II. iii) In GroEL that was spinlabeled at room temperature and subsequently used in HCA II binding experiments, the labels showed greater mobility in the presence of HCA II. iv) Fluoresceine-labeled GroEL displayed decreased fluorescence anisotropy, indicating higher flexibility during HCA II binding. This is qualitatively similar to results obtained from GroES binding, which has previously been shown to cause GroEL to expand. v) The quantum yield of AEDANS-labeled GroEL was increased in the presence of HCA II. All data indicated a more flexible and open structure of GroEL as a cause of binding HCA II, that can be utilized to further unfold the protein substrate. GroEL was, however, not capable to dissolve aggregates that were preformed from a molten-globule intermediate. Thus, it seems that the surface that is actively affected by GroEL must be exposed, and not hidden as in the HCA II aggregates, to allow GroEL to exert its chaperone activity. This suggests that surfaces responsible for off-pathway aggregation are overlapping with the surfaces involved in the interaction with chaperones.