Holo- and apo-bound structures of bacterial periplasmic heme-binding proteins

An essential component of heme transport in Gram-negative bacterial pathogens is the periplasmic protein that shuttles heme between outer and inner membranes. We have solved the first crystal structures of two such proteins, ShuT from Shigella dysenteriae and PhuT from Pseudomonas aeruginosa. Both share a common architecture typical of Class III periplasmic binding proteins. The heme binds in a narrow cleft between the N- and C-terminal binding domains and is coordinated by a Tyr residue. A comparison of the heme-free (apo) and -bound (holo) structures indicates little change in structure other than minor alterations in the heme pocket and movement of the Tyr heme ligand from an "in" position where it can coordinate the heme iron to an "out" orientation where it points away from the heme pocket. The detailed architecture of the heme pocket is quite different in ShuT and PhuT. Although Arg(228) in PhuT H-bonds with a heme propionate, in ShuT a peptide loop partially takes up the space occupied by Arg(228), and there is no Lys or Arg H-bonding with the heme propionates. A comparison of PhuT/ShuT with the vitamin B(12)-binding protein BtuF and the hydroxamic-type siderophore-binding protein FhuD, the only two other structurally characterized Class III periplasmic binding proteins, demonstrates that PhuT/ShuT more closely resembles BtuF, which reflects the closer similarity in ligands, heme and B(12), compared with ligands for FhuD, a peptide siderophore.     

Molecular dynamics simulations reveal that apo‐HisJ can sample a closed conformation

The Escherichia coli histidine binding protein HisJ is a type II periplasmic binding protein (PBP) that preferentially binds histidine and interacts with its cytoplasmic membrane ABC transporter, HisQMP₂, to initiate histidine transport. HisJ is a bilobal protein where the larger Domain 1 is connected to the smaller Domain 2 via two linking strands. Type II PBPs are thought to undergo “Venus flytrap” movements where the protein is able to reversibly transition from an open to a closed conformation. To explore the accessibility of the closed conformation to the apo state of the protein, we performed a set of all‐atom molecular dynamics simulations of HisJ starting from four different conformations: apo‐open, apo‐closed, apo‐semiopen, and holo‐closed. The simulations reveal that the closed conformation is less dynamic than the open one. HisJ experienced closing motions and explored semiopen conformations that reverted to closed forms resembling those found in the holo‐closed state. Essential dynamics analysis of the simulations identified domain closing/opening and twisting as main motions. The formation of specific inter‐hinge strand and interdomain polar interactions contributed to the adoption of the closed apo‐conformations although they are up to 2.5‐fold less prevalent compared with the holo‐closed simulations. The overall sampling of the closed form by apo‐HisJ provides a rationale for the binding of unliganded PBPs with their cytoplasmic membrane ABC transporters. Proteins 2014; 82:386–398. © 2013 Wiley Periodicals, Inc.     

Opening and closing motions in the periplasmic vitamin B12 binding protein BtuF

BtuF is the periplasmic binding protein (PBP) in the vitamin B(12) uptake system in Escherichia coli where it is associated with the ABC transporter BtuCD. When the ligand binds, PBPs generally display large conformational changes, commonly termed the Venus flytrap mechanism. BtuF belongs to a group of PBPs that, on the basis of crystal structures, does not appear to display such behavior. Using 480 ns multicopy molecular dynamics simulations of apo and holo forms of the protein, we investigate the dynamics of BtuF. We find BtuF to be more flexible than previously assumed, displaying clear opening and closing motions which are more pronounced in the apo form. The protein behavior is compatible with a PBP functional model that postulates a closed conformation for the ligand-bound state, whereas the empty form fluctuates between open and closed conformations. Elastic network normal-mode analysis suggests that all BtuF-like PBPs are capable of similar opening and closing motions. It also makes the typical Venus flytrap domain motions a likely common means of how PBP-ABC transporter interaction could occur.     

Two stacked heme molecules in the binding pocket of the periplasmic heme-binding protein HmuT from Yersinia pestis

The periplasmic binding protein HmuT from Yersinia pestis (YpHmuT) is a component of the heme uptake locus hmu and delivers bound hemin to the inner-membrane-localized, ATP-binding cassette (ABC) transporter HmuUV for translocation into the cytoplasm. The mechanism of this process, heme transport across the inner membrane of pathogenic bacteria, is currently insufficiently understood at the molecular level. Here we describe the crystal structures of the substrate-free and heme-bound states of YpHmuT, revealing two lobes with a central binding cleft. Superposition of the apo and holo states reveals a minor tilting motion of the lobes surrounding concomitant with heme binding. Unexpectedly, YpHmuT binds two stacked hemes in a central binding cleft that is larger than those of the homologous periplasmic heme-binding proteins ShuT and PhuT, both of which bind only one heme. The hemes bound to YpHmuT are coordinated via a tyrosine side chain that contacts the Fe atom of one heme and a histidine that contacts the Fe atom of the other heme. The coordinating histidine is only conserved in a subset of periplasmic heme binding proteins suggesting that its presence predicts the ability to bind two heme molecules simultaneously. The structural data are supported by spectroscopic binding studies performed in solution, where up to two hemes can bind to YpHmuT. Isothermal titration calorimetry suggests that the two hemes are bound in discrete, sequential steps and with dissociation constants (K_D) of ∼ 0.29  and ∼ 29 nM, which is similar to the affinities observed in other bacterial substrate binding proteins. Our findings suggest that the cognate ABC transporter HmuUV may simultaneously translocate two hemes per reaction cycle.     

Conformational and dynamics changes induced by bile acids binding to chicken liver bile acid binding protein

The correlation between protein motions and function is a central problem in protein science. Several studies have demonstrated that ligand binding and protein dynamics are strongly correlated in intracellular lipid binding proteins (iLBPs), in which the high degree of flexibility, principally occurring at the level of helix-II, CD, and EF loops (the so-called portal area), is significantly reduced upon ligand binding. We have recently investigated by NMR the dynamic properties of a member of the iLBP family, chicken liver bile acid binding protein (cL-BABP), in its apo and holo form, as a complex with two bile salts molecules. Binding was found to be regulated by a dynamic process and a conformational rearrangement was associated with this event. We report here the results of molecular dynamics (MD) simulations performed on apo and holo cL-BABP with the aim of further characterizing the protein regions involved in motion propagation and of evaluating the main molecular interactions stabilizing bound ligands. Upon binding, the root mean square fluctuation values substantially decrease for CD and EF loops while increase for the helix-loop-helix region, thus indicating that the portal area is the region mostly affected by complex formation. These results nicely correlate with backbone dynamics data derived from NMR experiments. Essential dynamics analysis of the MD trajectories indicates that the major concerted motions involve the three contiguous structural elements of the portal area, which however are dynamically coupled in different ways whether in the presence or in the absence of the ligands. Motions of the EF loop and of the helical region are part of the essential space of both apo and holo-BABP and sample a much wider conformational space in the apo form. Together with NMR results, these data support the view that, in the apo protein, the flexible EF loop visits many conformational states including those typical of the holo state and that the ligand acts stabilizing one of these pre-existing conformations. The present results, in agreement with data reported for other iLBPs, sharpen our knowledge on the binding mechanism for this protein family.     

Characterization of the periplasmic heme-binding protein shut from the heme uptake system of Shigella dysenteriae

The heme uptake systems by which bacterial pathogens acquire and utilize heme have recently been described. Such systems may utilize heme directly from the host's hemeproteins or via a hemophore that sequesters and transports heme to an outer membrane receptor and subsequently to the translocating proteins by which heme is further transported into the cell. However, little is known of the heme binding and release mechanisms that facilitate the uptake of heme into the pathogenic organism. As a first step toward elucidating the molecular level events that drive heme binding and release, we have undertaken a spectroscopic and mutational study of the first purified periplasmic heme-binding protein (PBP), ShuT from Shigella dysenteriae. On the basis of sequence identity, the ShuT protein is most closely related to the class of PBPs typified by the vitamin B(12) (BtuF) and iron-hydroxamate (FhuD) PBPs and is a monomeric protein having a molecular mass of 28.5 kDa following proteolytic processing of the periplasmic signaling peptide. ShuT binds one b-type heme per monomer with high affinity and bears no significant homology with other known heme proteins. The resonance Raman, MCD, and UV-visible spectra of WT heme-ShuT are consistent with a five-coordinate high spin heme having an anionic O-bound proximal ligand. Site-directed ShuT mutants of the absolutely conserved Tyr residues, Tyr-94 (Y94A) and Tyr-228 (Y228F), which are found in all putative periplasmic heme-binding proteins, were subjected to UV-visible, resonance Raman, and MCD spectroscopic investigations of heme coordination environment and rates of heme release. The results of these experiments confirmed Tyr-94 as the only axial heme ligand and Tyr-228 as making a significant contribution to the stability of heme-loaded ShuT, albeit without directly interacting with the heme iron.     

Substrate Binding Specifically Modulates Domain Arrangements in Adenylate Kinase

The enzyme adenylate kinase (ADK) features two substrate binding domains that undergo large-scale motions during catalysis. In the apo state, the enzyme preferentially adopts a globally open state with accessible binding sites. Binding of two substrate molecules (AMP + ATP or ADP + ADP) results in a closed domain conformation, allowing efficient phosphoryl-transfer catalysis. We employed molecular dynamics simulations to systematically investigate how the individual domain motions are modulated by the binding of substrates. Two-dimensional free-energy landscapes were calculated along the opening of the two flexible lid domains for apo and holo ADK as well as for all single natural substrates bound to one of the two binding sites of ADK. The simulations reveal a strong dependence of the conformational ensembles on type and binding position of the bound substrates and a nonsymmetric behavior of the lid domains. Altogether, the ensembles suggest that, upon initial substrate binding to the corresponding lid site, the opposing lid is maintained open and accessible for subsequent substrate binding. In contrast, ATP binding to the AMP-lid induces global domain closing, preventing further substrate binding to the ATP-lid site. This might constitute a mechanism by which the enzyme avoids the formation of a stable but enzymatically unproductive state.     

Dynamics of cellular retinoic acid binding protein I on multiple time scales with implications for ligand binding

Cellular retinoic acid binding protein I (CRABPI) belongs to the family of intracellular lipid binding proteins (iLBPs), all of which bind a hydrophobic ligand within an internal cavity. The structures of several iLBPs reveal minimal structural differences between the apo (ligand-free) and holo (ligand-bound) forms, suggesting that dynamics must play an important role in the ligand recognition and binding processes. Here, a variety of nuclear magnetic resonance (NMR) spectroscopy methods were used to systematically study the dynamics of both apo and holo CRABPI at various time scales. Translational and rotational diffusion constant measurements were used to study the overall motions of the proteins. Both apo and holo forms of CRABPI tend to self-associate at high (1.2 mM) concentrations, while at low concentrations (0.2 mM), they are predominantly monomeric. Rapid amide exchange rate and laboratory frame relaxation rate measurements at two spectrometer field strengths (500 and 600 MHz) were used to probe the internal motions of the individual residues. Several residues in the apo form, notably within the ligand recognition region, exhibit millisecond time scale motions that are significantly arrested in the holo form. In contrast, no significant differences in the high-frequency motions were observed between the two forms. These results provide direct experimental evidence for dynamics-induced ligand recognition and binding at a specifically defined time scale. They also exemplify the importance of dynamics in providing a more comprehensive understanding of how a protein functions.     

Biliverdin Reductase B Dynamics Are Coupled to Coenzyme Binding

Biliverdin reductase B (BLVRB) is a newly identified cellular redox regulator that catalyzes the NADPH-dependent reduction of multiple substrates. Through mass spectrometry analysis, we identified high levels of BLVRB in mature red blood cells, highlighting the importance of BLVRB in redox regulation. The BLVRB conformational changes that occur during conezyme/substrate binding and the role of dynamics in BLVRB function, however, remain unknown. Through a combination of NMR, kinetics, and isothermal titration calorimetry studies, we determined that BLVRB binds its coenzyme 500-fold more tightly than its substrate. While the active site of apo BLVRB is highly dynamic on multiple timescales, active site dynamics are largely quenched within holo BLVRB, in which dynamics are redistributed to other regions of the enzyme. We show that a single point mutation of Arg78?Ala leads to both an increase in active site micro-millisecond motions and an increase in the microscopic rate constants of coenzyme binding. This demonstrates that altering BLVRB active site dynamics can directly cause a change in functional characteristics. Our studies thus address the solution behavior of apo and holo BLVRB and identify a role of enzyme dynamics in coenzyme binding.     

Conformational dynamics of M2 helices in KirBac channels: helix flexibility in relation to gating via molecular dynamics simulations

KirBac1.1 and 3.1 are bacterial homologues of mammalian inward rectifier K channels. We have performed extended molecular dynamics simulations (five simulations, each of >20 ns duration) of the transmembrane domain of KirBac in two membrane environments, a palmitoyl oleoyl phosphatidylcholine bilayer and an octane slab. Analysis of these simulations has focused on the conformational dynamics of the pore-lining M2 helices, which form the cytoplasmic hydrophobic gate of the channel. Principal components analysis reveals bending of M2, with a molecular hinge at the conserved glycine (Gly134 in KirBac1.1, Gly120 in KirBac3.1). More detailed analysis reveals a dimer-of-dimers type motion. The first two eigenvectors describing the motions of M2 correspond to helix kink and swivel motions. The conformational flexibility of M2 seen in these simulations correlates with differences in M2 conformation between that seen in the X-ray structures of closed channels (KcsA and KirBac) in which the helix is undistorted, and in open channels (e.g. MthK) in which the M2 helix is kinked. Thus, the simulations, albeit on a time scale substantially shorter than that required for channel gating, suggest a gating model in which the intrinsic flexibility of M2 about a molecular hinge is coupled to conformational transitions of an intracellular 'gatekeeper' domain, the latter changing conformation in response to ligand binding.     

Molecular dynamics simulations of the conformational changes of the glutamate receptor ligand-binding core in the presence of glutamate and kainate

Excitatory synaptic transmission is mediated by ionotropic glutamate receptors (iGluRs) through the induced transient opening of transmembrane ion channels. The three-dimensional structure of the extracellular ligand-binding core of iGluRs shares the overall features of bacterial periplasmic binding proteins (PBPs). In both families of proteins, the ligand-binding site is arranged in two domains separated by a cleft and connected by two peptide stretches. PBPs undergo a typical hinge motion of the two domains associated with ligand binding that leads to a conformational change from an open to a closed form. The common architecture suggests a similar closing mechanism in the ligand-binding core of iGluRs induced by the binding of specific agonists. Starting from the experimentally determined kainate-bound closed form of the S1S2 GluR2 construct, we have studied by means of molecular dynamics simulations the opening motion of the ligand-binding core in the presence and in the absence of both glutamate and kainate. Our results suggest that the opening/closing interdomain hinge motions are coupled to conformational changes in the insertion region of the transmembrane segments. These changes are triggered by the interaction of the agonists with the essential Glu 209 residue. A plausible mechanism for the coupling of agonist binding to channel gating is discussed.     

Heme proteins—Diversity in structural characteristics,function, and folding

The characteristics of heme prosthetic groups and their binding sites have been analyzed in detail in a data set of nonhomologous heme proteins. Variations in the shape, volume, and chemical composition of the binding site, in the mode of heme binding and in the number and nature of heme–protein interactions are found to result in significantly different heme environments in proteins with different functions in biology. Differences are also seen in the properties of the apo states of the proteins. The apo states of proteins that bind heme permanently in their functional form show some disorder, ranging from local unfolding in the heme binding pocket to complete unfolding to give a random coil. In contrast, proteins that bind heme transiently are fully folded in their apo and holo states, presumably allowing both apo and holo forms to remain biologically active resisting aggregation or proteolysis. The principles identified here provide a framework for the design of de novo proteins that will exhibit tight heme ligand binding and for the identification of the function of structural genomic target proteins with heme ligands. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Effects of heme on the structure of the denatured state and folding kinetics of cytochrome b562

Heme-linked proteins, such as cytochromes, are popular subjects for protein folding studies. There is the underlying question of whether the heme affects the structure of the denatured state by cross-linking it and forming other interactions, which would perturb the folding pathway. We have studied wild-type and mutant cytochrome b562 from Escherichia coli, a 106 residue four-alpha-helical bundle. The holo protein apparently refolds with a half-life of 4 micros in its ferrous state. We have analysed the folding of the apo protein using continuous-flow fluorescence as well as stopped-flow fluorescence and CD. The apo protein folded much more slowly with a half-life of 270 micros that was unaffected by the presence of exogenous heme. We examined the nature of the denatured states of both holo and apo proteins by NMR methods over a range of concentrations of guanidine hydrochloride. The starting point for folding of the holo protein in concentrations of denaturant around the denaturation transition was a highly ordered native-like species with heme bound. Fully denatured holo protein at higher concentrations of denaturant consisted of denatured apo protein and free heme. Our results suggest that the very fast folding species of denatured holo protein is in a compact state, whereas the normal folding pathway from fully denatured holo protein consists of the slower folding of the apo protein followed by the binding of heme. These data should be considered in the analysis of folding of heme proteins.     

Substrate binding modifies the hinge bending characteristics of human 3‐phosphoglycerate kinase: A molecular dynamics study

3‐Phosphogycerate kinase (PGK) is a two domain enzyme, with a binding site of the 1,3‐bisphosphoglycerate on the N‐domain and of the ADP on the C‐domain. To transfer a phosphate group the enzyme has to undergo a hinge bending motion from open to closed conformation to bring the substrates to close proximity. Molecular dynamics simulation was used to elucidate the effect of ligand binding onto the domain motions of this enzyme. The simulation results of the apo form indicate a hinge bending motion in the ns timescale while the time period of the hinge bending motion of the complex form is clearly over the 20 ns simulation time. The apo form exhibits several hinge points that contribute to the hinge bending motion while upon binding the ligands, the hinge bending becomes strictly restrained with one dominant hinge point in the vicinity of the substrates. At the same time, ligand binding results in an enhanced correlation of internal domain motions. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Holo‐BtuF stabilizes the open conformation of the vitamin B12 ABC transporter BtuCD

While there is evidence that other ABC transporters can tell between empty and loaded substrate binding protein, reconstitution experiments suggest otherwise for the Escherichia coli vitamin B12 importer BtuCD‐F. Here, we address the question of BtuCD‐F substrate sensitivity in a combined protein–protein docking and molecular dynamics simulation approach. Starting from the BtuCD and holo‐BtuF crystal structures, we model two holo‐BtuCD‐F docking complexes differing by a 180° orientation of BtuF. One of these is similar to the apo‐BtuCD‐F crystal structure. Both docking complexes were embedded in a lipid/water environment to investigate their dynamics and BtuCD's conformational response to the presence and absence of BtuF, vitamin B12, and Mg‐ATP in a series of 28 independent MD simulations. We find holo‐BtuF stabilizing the open conformation of BtuCD, whereas the transporter begins to close again when BtuF or vitamin B12 is removed—suggesting BtuCD‐F is capable of substrate sensitivity. We identified BtuC transmembrane helices 3 and 5, the L‐loops and the adjacent helices comprised of BtuC residues 170–180 as hotspots of conformational change. We propose the latter to act as substrate sensors. BtuF‐Trp44 appears to act as a lid on the vitamin B12 binding cleft in BtuF X‐ray structures and protrudes into the BtuCD transport channel in one of our simulations, which might represent an initial step in vitamin B12 uptake. On an average, we observe subunit motions where the nucleotide binding domains approach each other while the transmembrane domains display an opening trend toward the periplasm. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Alternative structural state of transferrin. The crystallographic analysis of iron-loaded but domain-opened ovotransferrin N-lobe

Transferrins bind Fe3+ very tightly in a closed interdomain cleft by the coordination of four protein ligands (Asp60, Tyr92, Tyr191, and His250 in ovotransferrin N-lobe) and of a synergistic anion, physiologically bidentate CO32-. Upon Fe3+ uptake, transferrins undergo a large scale conformational transition: the apo structure with an opening of the interdomain cleft is transformed into the closed holo structure, implying initial Fe3+ binding in the open form. To solve the Fe3+-loaded, domain-opened structure, an ovotransferrin N-lobe crystal that had been grown as the apo form was soaked with Fe3+-nitrilotriacetate, and its structure was solved at 2.1 A resolution. The Fe3+-soaked form showed almost exactly the same overall open structure as the iron-free apo form. The electron density map unequivocally proved the presence of an iron atom with the coordination by the two protein ligands of Tyr92-OH and Tyr191-OH. Other Fe3+ coordination sites are occupied by a nitrilotriacetate anion, which is stabilized through the hydrogen bonds with the peptide NH groups of Ser122, Ala123, and Gly124 and a side chain group of Thr117. There is, however, no clear interaction between the nitrilotriacetate anion and the synergistic anion binding site, Arg121.     

Molecular dynamics simulations of the holo and apo forms of retinol binding protein. Structural and dynamical changes induced by retinol removal

The effects of removing retinol from the X-ray structure of holo-retinol binding protein are studied using the molecular dynamics technique. Structural and dynamical properties emerging from an 80 ps simulation of the apo form, for which no crystallographic structure is available, are compared with the results of a 70 ps trajectory of the holo-protein. Dynamical stationarity is attained after roughly 30 ps, and the resulting average structure is proposed as a reasonable model of the apo-protein. Conformational changes are observed for the loops at the beta-barrel entrance during the non-equilibrium part of the apo-trajectory. Tryptophan labelling experiments and retinoid reconstitution experiments point towards this part of the molecule as being involved in prealbumin binding. Structural changes in this region may therefore explain the differences in prealbumin affinity between the apo and holo forms. Furthermore, a change in the position of the alpha-helix, corresponding to a pivot around its C terminus, is observed for the apo-protein. The resulting conformation of the alpha-helix is found to be similar to that in apo-beta-lactoglobulin, which also can bind retinol and for which a crystal structure exists. The results from the holo simulation are compared to the crystallographic data and show good agreement. The dynamics of the secondary and tertiary structural elements are analysed and compared for the two forms. The beta-barrel is found to be extremely cooperative in its atomic motions in both simulations, and the top and bottom beta-sheets perform collective fluctuations with respect to each other in the low-frequency limit of the simulations. The dynamics of the alpha-helical region presents clear differences between the two forms; while the holo-protein has a well-defined spectrum for the longitudinal stretching mode, the apo form displays a fairly large bending of the alpha-helix at several points of the trajectory.     

Functional characterization of the Shigella dysenteriae heme ABC transporter

The heme ATP binding cassette (ABC) transporter, ShuUV, of Shigella dysenteriae has been incorporated into proteoliposomes. Functional characterization of ShuUV revealed that ATP hydrolysis and transport of heme from the periplasmic binding protein, ShuT, to the cytoplasmic binding protein, ShuS, are coupled. Site-directed mutagenesis of ShuT residues proposed to be required for stabilization of the complex abolished heme transport. Furthermore, residues His-252 and His-262, located in the translocation channel of ShuU, were required for the release of heme from ShuT and translocation to ShuS. The initial functional characterization of an in vitro heme uptake system provides a platform for future spectroscopic studies.     

Modulation of the structural integrity of helix F in apomyoglobin by single amino acid replacements

The conformational features of native and mutant forms of sperm-whale apomyoglobin (apoMb) at neutral pH were probed by limited proteolysis experiments utilizing up to eight proteases of different substrate specificities. It was shown that all proteases selectively cleave apoMb at the level of chain segment 82-94 (HEAELKPLAQSHA), encompassing helix F in the X-ray structure of the holo form of the native protein; for example, thermolysin cleaves the Pro 88-Leu 89 peptide bond. These results indicate that helix F is highly flexible or largely disrupted in apoMb. Because helix F contains the helix-breaking Pro 88 residue, we propose that helix F is kept in place in the native holo protein by a variety of helix-heme stabilizing interactions. To modulate the stability of helix F, the Pro88Ala and Pro88Gly mutants were prepared by site-directed mutagenesis, and their conformational properties investigated by both far-UV circular dichroism spectroscopy and limited proteolysis. The helix content of the Pro88Ala mutant was somewhat enhanced with respect to that of both native and Pro88Gly mutant, as expected from the fact that Ala is the strongest helix inducer among the 20 amino acid residues. The rate of limited proteolysis of the three apoMb variants by thermolysin and proteinase K was in the order native > Pro88Gly > Pro88Ala, in agreement with the scale of helix propensity of Ala, Gly, and Pro. The possible role of the flexible/unfolded chain segment 82-94 for the function and fate of apoMb at the cellular level is discussed.     

Molecular dynamics simulations of Hydrogenobacter thermophilus cytochrome c552: comparisons of the wild-type protein, a b-type variant, and the apo state

Molecular dynamic simulations have been performed for wild-type Hydrogenobacter thermophilus cytochrome c(552), a b-type variant of the protein, and the apo state with the heme prosthetic group removed. In the b-type variant, Cys 10 and Cys 13 were mutated to alanine residues, and so the heme group was no longer covalently bound to the protein. Two 8-ns simulations have been performed for each system at 298 and 360 K. The simulations of the wild-type protein at 298 K show a very close agreement with experimental NMR data. A fluxional process involving the side chain of Met 59, which coordinates to the heme iron, is observed in accord with proposals from NMR studies. Overall, the structure and dynamical behavior of the protein during the simulations of the b-type variant is closely similar to that of the wild-type protein. However, side chains in the heme-binding site show larger fluctuations in the b-type variant simulation at 360 K. In addition, structural changes are seen for a number of residues close to the heme group, particularly Gly 22 and Ser 51. The simulations of the apo state show significant conformational changes for residues 50-59. These residues form a loop region, which packs over the heme group in the wild-type protein and hydrogen bonds to the heme propionate groups. In the absence of heme, in the apo state simulations, these residues form short but persistent regions of beta-sheet secondary structure. These could provide nucleation sites for the conversion to amyloid fibrils.