Human prion protein: exploring the thermodynamic stability and structural dynamics of its pathogenic mutants.

Human familial prion diseases are known to be associated with different single-point mutants of the gene coding for prion protein with a primary focus at several locations of the globular domain. We have identified 12 different single-point pathogenic mutants of human prion protein (HuPrP) with the help of extensive perturbations/mutation technique at multiple locations of HuPrP sequence related to potentiality towards conformational disorders. Among these, some of the mutants include pathogenic variants that corroborate well with the literature reported proteins while majority include some unique single-point mutants that are either not explicitly studied early or studied for variants with different residues at the specific position. Primarily, our study sheds light on the unfolding mechanism of the above mentioned mutants in depth. Besides, we could identify some mutants under investigation that demonstrates not only unfolding of the helical structures but also extension and generation of the ß-sheet structures and or simultaneously have highly exposed hydrophobic surface which is assumed to be linked with the production of aggregate/fibril structures of the prion protein. Among the identified mutants, Q212E needs special attention due to its maximum exposure of hydrophobic core towards solvent and E200Q is found to be important due to its maximum extent of ß-content. We are also able to identify different respective structural conformations of the proteins according to their degree of structural unfolding and those conformations can be extracted and further studied in detail. Communicated by Ramaswamy H. Sarma.

Identification of putative unfolding intermediates of the mutant His-107-tyr of human carbonic anhydrase II in a multidimensional property space.

In this article, we develop an extensive search procedure of the multi-dimensional folding energy landscape of a protein. Our aim is to identify different classes of structures that have different aggregation propensities and catalytic activity. Following earlier studies by Daggett et al. [Jong, D. D.; Riley, R.: Alonso, D.O.: Dagett, V. J. Mol. Biol. 2002, 319, 229], a series of high temperature all-atom classical molecular simulation studies has been carried out to derive a multi-dimensional property space. Dynamical changes in these properties are then monitored by projecting them along a one-dimensional reaction coordinate, dmean . We have focused on the application of this method to partition a wide array of conformations of wild type human carbonic anhydrase II (HCA II) and its unstable mutant His-107-Tyr along dmean by sampling a 35-dimensional property space. The resultant partitioning not only reveals the distribution of conformations corresponding to stable structures of HCA II and its mutant, but also allows the monitoring of several partially unfolded and less stable conformations of the mutant. We have investigated the population of these conformations at different stages of unfolding and collected separate sets of structures that are widely separated in the property space. The dynamical diversity of these sets are examined in terms of the loading of their respective first principal component. The partially unfolded structures thus collected are qualitatively mapped on to the experimentally postulated light molten globule (MGL) and molten globule (MG) intermediates with distinct aggregation propensities and catalytic activities. Proteins 2016; 84:726-743. © 2016 Wiley Periodicals, Inc.

Modeling the structure and proton transfer pathways of the mutant His-107-Tyr of human carbonic anhydrase II.

We present molecular modeling of the structure and possible proton transfer pathways from the surface of the protein to the zinc-bound water molecule in the active site of the mutant His-107-Tyr of human carbonic anhydrase II (HCAII). No high-resolution structure or crystal structure is available till now for this particular mutant due to its lack of stability at physiological temperature. Our analysis utilizes as starting point a series of structures derived from high-resolution crystal structure of the wild type protein. While many of the structures investigated do not reveal a complete path between the zinc bound water and His-64, several others do indicate the presence of a transient connection even when His-64 is present in its outward conformation. Mutation at the residue 107 also reveals the formation of a new path into the active site. Competing contributions from His-64 sidechain rotation from its outward conformation are also evaluated in terms of optimal path analysis. No indication of a lower catalytic efficiency of the mutant is evident from our results under the condition of thermal stability of the mutant.

Measurement of distal histidine coordination equilibrium and kinetics in hexacoordinate hemoglobins.

The kinetics of ligand binding to hemoglobins has been measured for decades. Initially, these studies were confined to readily available pentacoordinate oxygen transport proteins like myoglobin, leghemoglobin, and red blood cell hemoglobin. Bimolecular ligand binding to these proteins is relatively simple, as ligand association is largely unimpeded at the heme iron. Although many techniques have been used to examine these reactions in the past, stopped-flow rapid mixing and flash photolysis are the most common ways to measure rate constants for ligand association and dissociation. Expression of recombinant proteins has allowed for examination of many newly discovered hemoglobins. The hexacoordinate hemoglobins are one such group of proteins that exhibit more complex binding kinetics than pentacoordinate hemoglobins due to reversible intramolecular coordination by a histidine side chain. Here, we describe methods for characterizing the kinetics of ligand binding to hexacoordinate hemoglobins with a focus on measurement of histidine coordination and exogenous ligand binding in both the ferrous and the ferric oxidation states.

Covalent heme attachment in Synechocystis hemoglobin is required to prevent ferrous heme dissociation.

Synechocystis hemoglobin contains an unprecedented covalent bond between a nonaxial histidine side chain (H117) and the heme 2-vinyl. This bond has been previously shown to stabilize the ferric protein against denaturation, and also to affect the kinetics of cyanide association. However, it is unclear why Synechocystis hemoglobin would require the additional degree of stabilization accompanying the His117-heme 2-vinyl bond because it also displays endogenous bis-histidyl axial heme coordination, which should greatly assist heme retention. Furthermore, the mechanism by which the His117-heme 2-vinyl bond affects ligand binding has not been reported, nor has any investigation of the role of this bond on the structure and function of the protein in the ferrous oxidation state. Here we report an investigation of the role of the Synechocystis hemoglobin His117-heme 2-vinyl bond on structure, heme coordination, exogenous ligand binding, and stability in both the ferrous and ferric oxidation states. Our results reveal that hexacoordinate Synechocystis hemoglobin lacking this bond is less stable in the ferrous oxidation state than the ferric, which is surprising in light of our understanding of pentacoordinate Hb stability, in which the ferric protein is always less stable. It is also demonstrated that removal of the His117-heme 2-vinyl bond increases the affinity constant for intramolecular histidine coordination in the ferric oxidation state, thus presenting greater competition for the ligand binding site and lowering the observed rate and affinity constants for exogenous ligands.

Influence of the protein matrix on intramolecular histidine ligation in ferric and ferrous hexacoordinate hemoglobins.

Present in most organisms, hexacoordinate hemoglobins (hxHbs) are proteins that have evolved the capacity for reversible bis-histidyl heme coordination. The heme prosthetic group enables diverse protein functionality, such as electron transfer, redox reactions, ligand transport, and enzymatic catalysis. The reactivity of heme is greatly effected by the coordination and noncovalent chemical environment imposed by its connate protein. Of considerable interest is how the hxHb globin fold achieves reversible intramolecular coordination while still enabling high-affinity binding of oxygen, nitric oxide, and other small ligands. Here we explore this question by examining the role of the protein matrix on coordination behavior in a group of hxHbs from animals, plants, and bacteria, including human neuroglobin and cytoglobin, a nonsymbiotic hemoglobin from rice, and a truncated hemoglobin from the cyanobacterium Synechocystis. This is done with a set of experiments measuring the reduction potentials of each wild-type hxHb and its corresponding mutant protein where the reversibly bound histidine (the distal His) has been replaced with a noncoordinating side chain. These reduction potentials, coupled with studies of the mutant proteins saturated with exogenous imidazole, enable us to assess the effects of the protein matrices on histidine coordination. Our results show significant variation among the hxHbs, demonstrating flexibility in the globin moiety's ability to regulate reversible coordination. This regulation is particularly evident in the plant nonsymbiotic hemoglobins, where ferric state histidine coordination affinity is substantially lowered by the protein matrix.

Role of phenylalanine B10 in plant nonsymbiotic hemoglobins.

All plants contain an unusual class of hemoglobins that display bis-histidyl coordination yet are able to bind exogenous ligands such as oxygen. Structurally homologous hexacoordinate hemoglobins (hxHbs) are also found in animals (neuroglobin and cytoglobin) and some cyanobacteria, where they are thought to play a role in free radical scavenging or ligand sensing. The plant hxHbs can be distinguished from the others because they are only weakly hexcacoordinate in the ferrous state, yet no structural mechanism for regulating hexacoordination has been articulated to account for this behavior. Plant hxHbs contain a conserved Phe at position B10 (Phe(B10)), which is near the reversibly coordinated distal His(E7). We have investigated the effects of Phe(B10) mutation on kinetic and equilibrium constants for hexacoordination and exogenous ligand binding in the ferrous and ferric oxidation states. Kinetic and equilibrium constants for hexacoordination and ligand binding along with CO-FTIR spectroscopy, midpoint reduction potentials, and the crystal structures of two key mutant proteins (F40W and F40L) reveal that Phe(B10) is an important regulatory element in hexacoordination. We show that Phe at this position is the only amino acid that facilitates stable oxygen binding to the ferrous Hb and the only one that promotes ligand binding in the ferric oxidation states. This work presents a structural mechanism for regulating reversible intramolecular coordination in plant hxHbs.