Faculty Summaries
Heinrich Roder, PhD
Heinrich Roder, PhD
Professor
  • Adjunct Professor, University of Pennsylvania
  • Professor, Temple University
  • Director, Spectroscopy Support Facility
Heinrich.Roder@fccc.edu
Office Phone: 215-728-3123
Fax: 215-728-3574
Office: R414
Lab: R407/408
  • Folding mechanism of reduced cytochrome c in the presence of carbon monoxide
    Structural changes in cytochrome c upon CO binding to the heme
    Structural changes in cytochrome c upon CO binding to the heme

    Despite close structural similarity, the ferric and ferrous forms of cytochrome c (cyt c) differ greatly in terms of their ligand binding properties, stability, folding and dynamics. The reduced heme iron binds diatomic ligands such as CO only under destabilizing conditions that promote weakening or disruption of the native methionine-iron linkage. This makes CO a useful conformational probe for detecting partially structured states that cannot be observed in the absence of endogenous ligands. We characterized the denaturant-induced unfolding equilibrium of reduced cyt c in the presence and absence of CO by means of heme absorbance, circular dichroism and NMR [Latypov et al, 2008]. In addition to the native state (N), which does not bind CO, and the unfolded CO-complex (U-CO), a structurally distinct CO-bound form (M-CO) accumulates to high levels at intermediate guanidine hydrochloride concentrations. Comparison of the unfolding transition for different conformational probes reveals that M-CO is a compact state containing a native-like helical core and regions of local disorder in the segment containing the native Met80 ligand and adjacent loops (Figure: Changes in the structure of cytochrome c due to binding of a carbon monoxide ligand to the heme). Kinetic measurements of CO binding and dissociation under native, partially denaturing and fully unfolded conditions indicate that a state, M, that is structurally analogous to M-CO is populated even in the absence of CO. The binding energy of the CO ligand lowers the free energy of this high-energy state to such an extent that it accumulates even under mildly denaturing equilibrium conditions. The thermodynamic and kinetic parameters obtained in this study provide a fully self-consistent description of the linked unfolding/CO-binding equilibria of reduced cyt c.

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  • Interplay between His-heme loop formation and oligomerization of denatured cyt c
    Kinetics of loop formation in unfolded cytochrome c
    Kinetics of loop formation in unfolded cytochrome c

    The kinetics of loop formation between His side chains and the covalently bound heme of cyt c have long been recognized as a unique source of information on the conformational and dynamic properties of denatured proteins. In collaboration with Bruce Bowler (University of Montana), we focused on the effect of Pro25 in yeast iso-1 cyt c on the formation of a relatively short loop between His26 and the heme [Tzul et al, 2009]. We measured the effect of protein concentration on the apparent pKa for His-heme binding and on the kinetics loop formation under denaturing conditions (3 M guanidine HCl) for variants containing Pro or Ala at position 25 (Figure: Kinetics of loop formation in unfolded cytochrome c). The results showed that the presence of a Pro, which promotes trans-cis peptide bond isomerization, has a major effect on the equilibrium and kinetics for forming the sterically constrained His26-heme loop. The Pro-containing variant has a strongly enhanced tendency for reversible oligomer formation. Kinetic modeling of the results provide quantitative insight into the coupling between folding and aggregation of denatured proteins, which is of considerable interest with respect to protein mis-folding/aggregation diseases.

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  • Folding kinetics of staphylococcal nuclease studied by tryptophan engineering and rapid mixing methods

    Elucidating the structural characteristics of transition state ensembles and transient intermediates in protein folding reactions is critical for understanding the mechanisms of protein folding. Recent advances in ultrarapid mixing and detection methods have made it possible to extend fluorescence- and absorbance-detected measurements of solvent-induced refolding reactions into the microsecond time regime, which has yielded a wealth of new insight into early stages of folding [Roder et al, 2006]. However, detailed structural information on these intermediates, especially with respect to tertiary interactions, is still lacking.

    Residues in staphylococcal nuclease targeted for Trp substitution
    Residues in staphylococcal nuclease targeted for Trp substitution

    To probe early structural events during folding of staphylococcal nuclease (SNase), a 149-residue protein comprising an β-barrel domain and a C-terminal cluster of α-helices, we are combining protein engineering with fluorescence-detected rapid mixing methods. Our paper in J. Mol. Biol. [Maki et al, 2007] describes the equilibrium fluorescence properties and kinetic folding mechanism of a series of single-Trp variants containing a unique tryptophan residue at various locations (residues 15, 27, 61, 76, 91, 102 and 121) throughout the structure of a tryptophan-free variant of staphylococcal nuclease (Figure: Residues in staphylococcal nuclease targeted for Trp substitution). Thermal unfolding measurements by circular dichroism indicate that the variants are destabilized, but maintain the ability to fold into a native-like structure. For the variants with Trp at positions 15, 27 and 61, the intrinsic fluorescence is significantly quenched in the native state due to close contact with polar side chains that act as intramolecular quenchers. All other variants exhibited enhanced fluorescence under native conditions consistent with burial of the tryptophans in an apolar environment. The kinetics of folding was observed by continuous- and stopped-flow fluorescence measurements over refolding times ranging from 100 μs to 10 s. The folding kinetics of all variants is quantitatively described by a mechanism involving a major pathway with a series of intermediate states and a minor parallel channel. The engineered tryptophans in the β-barrel and the N-terminal part of the α-helical domain become partially shielded from the solvent at an early stage (< 1 ms), indicating that this region of the protein undergoes a rapid specific collapse and remains uncoupled from the rest of the α-helical domain until the late stages of folding. For several variants, a major increase in fluorescence coincides with the rate-limiting step of folding on the 100 ms time scale, indicating that these tryptophans reach their buried native environment only during the late stages of folding. Other variants show more complex behavior with a transient increase in fluorescence during the 10 ms phase followed by a decrease during the rate-limiting phase. These observations are consistent with burial of these probes in a collapsed, but loosely packed intermediate, followed by the rate-limiting formation of the densely packed native core, which brings the tryptophans into close contact with intramolecular quenchers.

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  • Formation of hydrogen-bonded structure during early stages of protein folding
    Capillary mixer for ultrafast quenched-flow H/D exchange experiments
    Capillary mixer for ultrafast quenched-flow H/D exchange experiments

    A major ongoing effort of our group is to gain detailed structural insight into early stages of folding of two model proteins, staphylococcal nuclease (SNase) and cytochrome c (cyt c) by combining ultra-rapid mixing techniques with hydrogen-deuterium exchange and NMR. This work capitalizes on the previous development of a capillary mixer for quenched-flow experiments with an unprecedented time resolution of 60 μs [reviewed in Roder et al, 2006]. In a typical H/D exchange labeling experiment, the protein is initially unfolded in D2O and rapidly mixed with an H2O refolding buffer at alkaline pH, which sets up a competition between refolding and solvent (H/D) exchange (Figure: Capillary mixer for ultrafast quenched-flow H/D exchange experiments). After a brief delay (typically 150 μs) the exchange reaction is quenched by injecting the solution into buffer at acidic pH, which effectively quenches the H/D exchange reaction while allowing the refolding reaction to go to completion. We recently employed this novel method to detect the formation of individual hydrogen bonds in an early folding intermediates of cyt c, which is formed in an initial folding phase with a time constant of 60 μs. About a dozen samples were collected at different labeling pH ranging from 7.5 to 12, and the proton occupancies of individual NH groups were measured by recording homonulcear 2D NMR spectra. By comparing the pH-dependence of the proton occupancies with those measured under denaturing conditions, one can thus characterize very short-lived folding intermediates in terms of the formation of individual hydrogen bonds. Clusters of amide protons in each of three α-helices of cyt c show substantial protection from solvent exchange, indicating that the initial compaction of the protein involves specific structure formation rather than a non-specific hydrophobic collapse. In a parallel study on SNase, the protein was again subjected to H/D exchange labeling at 150 μs while undergoing refolding and 2D 1H-15N correlation spectra were collected for a series of samples subjected to H/D exchange labeling at 15 different pH values between about 7.5 and 12.5. The data show surprisingly large variation in protection behavior for different amid protons, indicating that the earliest fluorescence-detected intermediate already contains hydrogen-bonded structure in several regions of the protein, including parts of the β-barrel as well as some helical segments.

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  • Early stages of folding of apomyoglobin

    The structural properties and significance of early intermediates in protein folding remain poorly understood. In particular, the question whether the conformational events that are often observed on a microsecond time scale represent bono fide folding events or kinetic traps remains controversial. In this project, we study early events in folding of apomyoglobin (apoMb), an α-helical protein known to fold via a series of partially structured states. By combining spectroscopic and kinetics techniques with protein engineering, we can identify key residues involved in stabilizing the partially folded forms and transition-state ensembles populated along the pathway of folding.

    Unfolding of WT apomyoglobin (left) and L115A mutant (right) at pH 4.0
    Unfolding of WT apomyoglobin (left) and L115A mutant (right) at pH 4.0

    We began by collecting a series of equilibrium urea unfolding data on a series of apoMb variants in which hydrophobic residues at critical helix-helix contact sites were replaced by both smaller and larger side chains. Tryptophan fluorescence emission spectra as a function of denaturant concentration were analyzed globally on the basis of a four-state unfolding mechanism, N ⇔ M ⇔ I ⇔ U, where N is the folded state of apoMb at pH 6.2, M is the predominant folded state at pH 4, I is a less structured equilibrium state observed at pH 4 and U is the fully unfolded state. The figure to the left shows representative global fitting results for wild type and L115A apoMb at pH 4. The complex wavelength-dependent unfolding transitions for these two proteins (as well as other variants) provide clear evidence for the presence of an equilibrium intermediate. The L115A mutation (decreasing hydrophobicity) preferentially destabilizes the M-state, resulting in an increased population for the I-state. In contrast, the M131W mutation (decreasing hydrophobicity) preferentially stabilizes the I-state. Qualitatively, the effects of mutations on the unfolding equilibrium at pH 4 suggest that the M-state, and especially the I-state, are stabilized by hydrophobic interactions between helices driven largely by hydrophobicity rather than specific side-chain interactions.

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  • Intrinsic disorder and flexible linkers in a signaling protein containing modular domains
    Domain structure of NHERF1 and 3D structure of PDZ domains
    Domain structure of NHERF1 and 3D structure of PDZ domains
    Domain structure of NHERF1 and 3D structure of PDZ domains
    Domain structure of NHERF1 and 3D structure of PDZ domains

    Many proteins contain extensive disordered regions or remain fully unfolded even under physiological conditions. Intrinsic disorder is especially common in cell-signaling proteins where individual structured domains are often interrupted by long disordered segments. In this project, we make use of various biophysical approaches, including NMR, thermodynamic and computational methods, in order to explore the structural and dynamic properties of the flexible linkers in Na+/H+ exchanger regulatory factor (NHERF1), a 358-residue signaling adaptor protein comprised of two globular domains belonging to the PSD-90/Dlg/ZO-1 (PDZ) domain family, as well as a C-terminal ezrin binding (EB) motif (Fig. 6). The ~100 residue segment separating the second PDZ domain from the EB motif at the C-terminus is predicted to be intrinsically disordered, using various disorder prediction algorithms. Nevertheless, we obtained clear evidence that the two domains can engage in specific intramolecular interactions, based on NMR chemical shift and relaxation dispersion measurements and equilibrium unfolding experiments involving circular dichroism or fluorescence [Cheng, Li, Fazlieva, Dai, Bu and Roder, Structure, in press]. The changes in NMR relaxation parameters and chemical shifts at the C-terminus of NHERF observed upon binding indicate that the initially disordered EB region becomes helical when it binds to PDZ2. This binding mode is highly unusual as PDZ domains generally recognize a few residues at the C-terminus of a protein in an extended conformation. Equilibrium unfolding measurements on mutant proteins with helix-disrupting (Gly or Pro) or promoting (Ala) mutations at solvent-exposed positions of the predicted α-helix corroborated the NMR data. Thermodynamic analysis of intra- and intermolecular PDZ2-EB interactions further revealed that an intrinsically disordered linker connecting the PDZ2 and EB regions contributes significantly to stabilizing the closed conformation of NHERF. The result is a complex unfolding equilibrium involving three structurally distinct folded states in addition to the fully unfolded state (Fig. 7). Only one of these, Iopen, binds a physiological ligand of NHERF1, whereas the binding site on PDZ2 is occluded in the more stable folded states, IEB and N, due to intramolecular interactions with CT. These dynamic contacts involving the C-terminal EB helix or the intervening flexible linker region have a profound effect on the overall structure of NHERF1 and modulate the binding properties of its domains, and thus are critical for understanding its functions as a signaling adaptor. Together with the fact that the linker sequence contains several potential phosphorylation sites, these observations suggest a novel functional role for intrinsically disordered protein segments in regulating the balance between intra- and inter-molecular ligand interactions.

    Figure 7
    Figure 7
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  • Role of early kinetic intermediates in folding and aggregation of prion proteins

    A number of diseases can be linked to the deposition of fibrillar aggregates of misfolded proteins or peptides in and around cells. In addition to several neurodegenerative and amyloid deposition diseases, protein aggregation also plays a critical role in transmissible spongiform encephalopathies, a family of infectious diseases in humans and animals caused by improper folding and self-association of the prion protein, PrPc. A rational approach to treating these diseases will require a more detailed understanding of the structural principles underlying the conversion of the normal cellular form of the prion protein, PrPc, into its infectious “scrapie” form, PrPSc, which forms neurotoxic aggregates with amyloid-like properties. PrPc is a 231-residue protein consists of a highly flexible N-terminal region and a globular C-terminal domain (residues 125-231) comprising three α-helices and two short β-strands. Although they are identical in terms of covalent structure, PrPc and PrPSc exhibit profoundly different biophysical properties. While PrPc is monomeric, protease-sensitive and soluble in aqueous buffer, PrPSc exists as a large, β-sheet containing, oligomer, is protease-resistant and insoluble.

    Rapid formation of an intermediate during folding of the human prion protein
    Rapid formation of an intermediate during folding of the human prion protein

    An important step towards understanding the mechanism of the PrPc to PrPSc conversion is to elucidate the folding pathways of the prion protein. Based on previous stopped-flow measurements, it has been proposed that the prion protein folds via a transient intermediate formed on the millisecond time-scale or faster, and mutations linked to familial diseases result in a pronounced increase in the population of this intermediate. In collaboration with Witold Surevicz at Case Western, we have extended these studies into the microsecond time scale using a capillary mixing system with a time resolution of less than 100 μs [Apetri et al., 2006]. This allowed us to directly observe two distinct phases in folding of the recombinant human prion protein (fragment 90-231 comprising the structured C-terminal domain and an unstructured N-terminal tail). The kinetic data obtained (Figure: Kinetic evidence for rapid formation of an early intermediate during folding of the human prion protein, PrP) provide clear evidence for rapid accumulation of an early intermediate (with a time constant of ~50 μs), followed by a rate-limiting folding step (with a time constant of ~700 μs). We further found that the population of the intermediate increases significantly at mildly acidic pH and in the presence of urea. Three-state folding behavior with enhanced population of an intermediate was also observed for a PrP variant harboring a F198S mutation, which is associated with a human neurodegenerative disease (Gerstmann-Straussler-Scheinker disease). Our findings strongly suggest that the partially structured intermediate detected in our kinetic experiments represents a monomeric precursor of the pathogenic PrPSc oligomer.

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  • Conformational changes associated with activation of blood coagulation factor XI studied by NMR structural analysis, solution scattering and EM
    Conformational changes associated with zymogen activation of factor XI
    Conformational changes associated with zymogen activation of factor XI

    We continue to collaborate with Peter N. Walsh at Temple University on structure-function studies of blood-coagulation factor XI, a 160 kDa dimeric protein comprising four N-terminal regulatory domains (A1 – A4) and a chimotrypsin-like catalytic domain. Inhibitors of factor XI are being developed as potential antithrombotic drugs. In a recent report in Proc. Natl. Acad. Sci. [Samuel et al, 2007], we used solution NMR methods to determine the 3D structure of the A4 domain of factor XI, which mediates formation of a disulfide-linked dimer. The core of the A4 domain consists of a central five-stranded β-sheet flanked by an α-helix on one side and a two-stranded β-sheet on the other. In contrast to the crystal structure of the full-length factor XI zymogen, the solution structure of the isolated A4 domain exhibits a second α-helix at the C-terminus. Combined with molecular modeling, this finding suggests that this helix is formed only upon activation of factor XI, leading to reorientation of the catalytic domains that would bring the two active sites into close proximity (Fig. 9). In direct support of this model, small-angle X-ray scattering and electron microscopic data reveal that activation of factor XI (triggered by cutting of the protein chain at a particular site) is accompanied by a significant change in overall shape, which modulates its enzymatic activity and binding properties. These findings provide a structural framework for understanding the activity of factor XI in blood clotting and serves as a paradigm for understanding the importance of protein structural rearrangements in regulating protein interactions and enzymatic activity. Our study for the first time offers a detailed structural explanation of activation for any blood coagulation protein.

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  • Dimer Dissociation and Unfolding Mechanism of Coagulation Factor XI Apple 4 Domain

    The blood coagulation protein factor XI (FXI) consists of a pair of disulfide-linked chains each containing four apple domains (A1-A4) and a trypsin-like serine protease domain. In response to blood vessel injury, the FXI zymogen is converted into its proteolytically active form, factor XIa (FXIa) through cleavage of a scissile bond (Arg369-Ile370). The enzymatic activators of FXI are thrombin and factor XIIa, and the physiological substrate of FXIa is factor IX. FXIa is part of a feedback loop, referred to as the consolidation phase of blood coagulation, resulting from activation of FXI by the small quantities of thrombin that are generated by the tissue factor pathway prior to its inhibition by tissue factor pathway inhibitor.

    Dimeric A4 dimerization domain of factor FXI
    Dimeric A4 dimerization domain of factor FXI

    The apple 4 domain of FXI (F272-E362) mediates noncovalent homodimer formation even when the cysteine involved in an intersubunit disulfide is mutated to serine (C321S). To understand the role of noncovalent interactions in stabilizing the FXI dimer, we monitored the equilibrium unfolding transitions of wild-type A4 and its C321S variant by circular dichroism (CD), intrinsic tyrosine fluorescence and dynamic light scattering measurements as a function of guanidine hydrochloride concentration [Riley et al, 2007 ]. Global analysis of the unimolecular unfolding transition of wild-type A4 revealed a partially unfolded equilibrium intermediate at low to moderate denaturant concentrations. The optically detected equilibrium of C321S A4 also fits best to a three-state model in which the native dimer unfolds via a monomeric intermediate state. Dimer dissociation is characterized by a dissociation constant, Kd, of ~90 nM (in terms of monomer) consistent with the dissociation constant measured independently using fluorescence anisotropy. The results imply that FXI folding occurs via a monomeric equilibrium intermediate. This observation sheds light on the effect of certain naturally occurring mutations, such as F283L, which lead to intracellular accumulation of non-native forms of FXI. To investigate the structural and energetic consequences of the F283L mutation, which perturbs a cluster of aromatic side chains within the core of the A4 monomer, it was introduced into the dissociable dimer, C321S A4. NMR chemical shift analysis confirmed that the mutant can assume a native-like dimeric structure (Figure a). However, equilibrium unfolding measurements show that the mutation causes a four-fold increase in the Kd for dissociation of the native dimer and a 1 kcal/mol stabilization of the monomer, resulting in a highly populated intermediate. Since the F283 side chain does not directly participate in the dimer interface, we propose that the F283L mutation leads to increased dimer dissociation by stabilizing a monomeric state with altered side chain packing that is unfavorable for homodimer formation (Figure b).

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