Proton Occupancies in Histidine Side Chains of Carbonic Anhydrase II by Neutron Crystallography and NMR - Differences, Similarities and Opportunities.

Histidine is a key amino-acid residues in proteins that can exist in three different protonation states: two different neutral tautomeric forms and a protonated, positively charged one. It can act as both donor and acceptor of hydrogen bonds, coordinate metal ions, and engage in acid/base catalysis. Human Carbonic Anhydrase II (HCA II) is a pivotal enzyme catalyzing the reversible hydration of carbon dioxide.

Proton Transfer Kinetics in Histidine Side Chains Determined by pH-Dependent Multi-Nuclear NMR Relaxation.

Histidine is a key amino-acid residue in proteins with unique properties engendered by its imidazole side chain that can exist in three different states: two different neutral tautomeric forms and a protonated, positively charged one with a p value close to physiological pH. Commonly, two or all three states coexist and interchange rapidly, enabling histidine to act as both donor and acceptor of hydrogen bonds, coordinate metal ions, and engage in acid/base catalysis. Understanding the exchange dynamics among the three states is critical for assessing histidine's mechanistic role in catalysis, where the rate of proton exchange and interconversion among tautomers might be rate limiting for turnover.

Energetics and dynamics of the proton shuttle of carbonic anhydrase II.

Human carbonic anhydrase II catalyzes the reversible reaction of carbon dioxide and water to form bicarbonate and a proton. His64-mediated proton shuttling between the active site and the bulk solvent is rate limiting. Here we investigate the protonation behavior of His64 as well as its structural and dynamic features in a pH dependent way.

Early Stage UV-B Induced Molecular Modifications of Human Eye Lens γD-Crystallin.

In the human eye lenses, the crystallin proteins facilitate transparency, light refraction, as well as UV light protection. A deregulated balanced interplay between α-, β-, and γ-crystallin can cause cataract. γD-crystallin (hγD) is involved in the energy dissipation of absorbed UV light by energy transfer between aromatic side chains.

NMR Studies of Aromatic Ring Flips to Probe Conformational Fluctuations in Proteins.

Aromatic residues form a significant part of the protein core, where they make tight interactions with multiple surrounding side chains. Despite the dense packing of internal side chains, the aromatic rings of phenylalanine and tyrosine residues undergo 180° rotations, or flips, which are mediated by transient and large-scale "breathing" motions that generate sufficient void volume around the aromatic ring. Forty years after the seminal work by Wagner and Wüthrich, NMR studies of aromatic ring flips are now undergoing a renaissance as a powerful means of probing fundamental dynamic properties of proteins.

Characterizing Fast Conformational Exchange of Aromatic Rings Using Residual Dipolar Couplings: Distinguishing Jumplike Flips from Other Exchange Mechanisms.

Aromatic ring flips are a hallmark of protein dynamics. They are experimentally studied by NMR spectroscopy, where recent advances have led to improved characterization across a wide range of time scales. Results on different proteins have been interpreted as continuous diffusive ring rotations or jumplike flips, leading to diverging views of the protein interior as being fluidlike or solidlike, respectively.

Impact of distant peptide substrate residues on enzymatic activity of SlyD.

Peptidyl-prolyl isomerases (PPIases) catalyze intrinsically slow and often rate-limiting isomerization of prolyl-peptide bonds in unfolded or partially folded proteins, thereby speeding up the folding process and preventing misfolding. They often possess binding and chaperone domains in addition to the domain carrying the isomerization activity. Although generally, their substrates display no identity in their amino acid sequence upstream and downstream of the proline with 20 possibilities for each residue, PPIases are efficient enzymes.

Monitoring protein unfolding transitions by NMR-spectroscopy.

NMR-spectroscopy has certain unique advantages for recording unfolding transitions of proteins compared e.g. to optical methods.

Completing the family of human Eps15 homology domains: Solution structure of the internal Eps15 homology domain of γ-synergin.

Eps15 homology (EH) domains are universal interaction domains to establish networks of protein-protein interactions in the cell. These networks mainly coordinate cellular functions including endocytosis, actin remodeling, and other intracellular signaling pathways. They are well characterized in structural terms, except for the internal EH domain from human γ-synergin (EHγ).

Macromolecular Crowding Induces a Binding Competent Transient Structure in Intrinsically Disordered Gab1.

Intrinsically disordered proteins (IDPs) are an important class of proteins which lack tertiary structure elements. Their dynamic properties can depend on reversible post-translational modifications and the complex cellular milieu, which provides a crowded environment. Both influences the thermodynamic stability and folding of globular proteins as well as the conformational plasticity of IDPs.

H R relaxation dispersion experiments in aromatic side chains.

Aromatic side chains are attractive probes of protein dynamic, since they are often key residues in enzyme active sites and protein binding sites. Dynamic processes on microsecond to millisecond timescales can be studied by relaxation dispersion experiments that attenuate conformational exchange contributions to the transverse relaxation rate by varying the refocusing frequency of applied radio-frequency fields implemented as either CPMG pulse trains or continuous spin-lock periods. Here we present an aromatic H R relaxation dispersion experiment enabling studies of two to three times faster exchange processes than achievable by existing experiments for aromatic side chains.

Transition-State Compressibility and Activation Volume of Transient Protein Conformational Fluctuations.

Proteins are dynamic entities that intermittently depart from their ground-state structures and undergo conformational transitions as a critical part of their functions. Central to understanding such transitions are the structural rearrangements along the connecting pathway, where the transition state plays a special role. Using NMR relaxation at variable temperature and pressure to measure aromatic ring flips inside a protein core, we obtain information on the structure and thermodynamics of the transition state.

Structural role of essential light chains in the apicomplexan glideosome.

Gliding, a type of motility based on an actin-myosin motor, is specific to apicomplexan parasites. Myosin A binds two light chains which further interact with glideosome associated proteins and assemble into the glideosome. The role of individual glideosome proteins is unclear due to the lack of structures of larger glideosome assemblies.

Ultrastructural evidence for self-replication of Alzheimer-associated Aβ42 amyloid along the sides of fibrils.

The nucleation of Alzheimer-associated Aβ peptide monomers can be catalyzed by preexisting Aβ fibrils. This leads to autocatalytic amplification of aggregate mass and underlies self-replication and generation of toxic oligomers associated with several neurodegenerative diseases. However, the nature of the interactions between the monomeric species and the fibrils during this key process, and indeed the ultrastructural localization of the interaction sites have remained elusive.

Slow ring flips in aromatic cluster of GB1 studied by aromatic C relaxation dispersion methods.

Ring flips of phenylalanine and tyrosine are a hallmark of protein dynamics. They report on transient breathing motions of proteins. In addition, flip rates also depend on stabilizing interactions in the ground state, like aromatic stacking or cation-π interaction.

Adsorption of unfolded Cu/Zn superoxide dismutase onto hydrophobic surfaces catalyzes its formation of amyloid fibrils.

Intracellular aggregates of superoxide dismutase 1 (SOD1) are associated with amyotrophic lateral sclerosis. In vivo, aggregation occurs in a complex and dense molecular environment with chemically heterogeneous surfaces. To investigate how SOD1 fibril formation is affected by surfaces, we used an in vitro model system enabling us to vary the molecular features of both SOD1 and the surfaces, as well as the surface area.

Rotamer Jumps, Proton Exchange, and Amine Inversion Dynamics of Dimethylated Lysine Residues in Proteins Resolved by pH-Dependent H and C NMR Relaxation Dispersion.

Post-translational methylation of lysine side chains is of great importance for protein regulation, including epigenetic control. Here, we present specific CHD labeling of dimethylated lysines as a sensitive probe of the structure, interactions, and dynamics of these groups, and outline a theoretical and experimental framework for analyzing their conformational dynamics using H and C CPMG relaxation dispersion experiments. Dimethylated lysine side chains in calcium-loaded calmodulin show a marked pH dependence of their Carr-Purcell-Meiboom-Gill (CPMG) dispersion profiles, indicating complex exchange behavior.

Site-selective H/H labeling enables artifact-free H CPMG relaxation dispersion experiments in aromatic side chains.

Aromatic side chains are often key residues in enzyme active sites and protein binding sites, making them attractive probes of protein dynamics on the millisecond timescale. Such dynamic processes can be studied by aromatic C or H CPMG relaxation dispersion experiments. Aromatic H CPMG relaxation dispersion experiments in phenylalanine, tyrosine and the six-ring moiety of tryptophan, however, are affected by J H-H couplings which are causing anomalous relaxation dispersion profiles.

Reprint of "Ganglioside lipids accelerate α-synuclein amyloid formation".

The deposition of α-synuclein fibrils is one hallmark of Parkinson's disease. Here, we investigate how ganglioside lipids, present in high amounts in neurons and exosomes, influence the aggregation kinetics of α-synuclein. Gangliosides, as well as, other anionic lipid species with small or large headgroups were found to induce conformational changes of α-synuclein monomers and catalyse their aggregation at mildly acidic conditions.

Optimal Isotope Labeling of Aromatic Amino Acid Side Chains for NMR Studies of Protein Dynamics.

Aromatic side chains in proteins are often directly evolved in stabilizing the hydrophobic core, protein binding, or enzymatic activity. They are also responsible for specific local dynamic processes, such as histidine tautomerization or ring flips. Despite their importance, they are often not targeted directly by NMR spectroscopy, because of spectroscopic complications and challenges.

Experimental pK Value Determination of All Ionizable Groups of a Hyperstable Protein.

Electrostatic interactions significantly contribute to the stability and function of proteins. The stabilizing or destabilizing effect of local charge is reflected in the perturbation of the pK value of an ionizable group from the intrinsic pK value. Herein, the charge network of a hyperstable dimeric protein (ribbon-helix-helix (rhh) protein from plasmid pRN1 from Sulfolobus islandicus) is studied through experimental determination of the pK values of all ionizable groups.

Conformational exchange of aromatic side chains by H CPMG relaxation dispersion.

Aromatic side chains are attractive probes of protein dynamics on the millisecond time scale, because they are often key residues in enzyme active sites and protein binding sites. Further they allow to study specific processes, like histidine tautomerization and ring flips. Till now such processes have been studied by aromatic C CPMG relaxation dispersion experiments.

Equilibrium and Kinetic Unfolding of GB1: Stabilization of the Native State by Pressure.

NMR spectroscopy allows an all-atom view on pressure-induced protein folding, separate detection of different folding states, determination of their population, and the measurement of the folding kinetics at equilibrium. Here, we studied the folding of protein GB1 at pH 2 in a temperature and pressure dependent way. We find that the midpoints of temperature-induced unfolding increase with higher pressure.

Ganglioside lipids accelerate α-synuclein amyloid formation.

The deposition of α-synuclein fibrils is one hallmark of Parkinson's disease. Here, we investigate how ganglioside lipids, present in high amounts in neurons and exosomes, influence the aggregation kinetics of α-synuclein. Gangliosides, as well as, other anionic lipid species with small or large headgroups were found to induce conformational changes of α-synuclein monomers and catalyse their aggregation at mildly acidic conditions.

Conserved S/T Residues of the Human Chaperone DNAJB6 Are Required for Effective Inhibition of Aβ42 Amyloid Fibril Formation.

The human molecular chaperone DNAJB6, an oligomeric protein with a conserved S/T-rich region, is an efficient suppressor of amyloid fibril formation by highly aggregation-prone peptides such as the Aβ and polyQ peptides associated with Alzheimer's and Huntington's disease, respectively. We previously showed that DNAJB6 can inhibit the processes through which amyloid fibrils are formed via strong interactions with aggregated forms of Aβ42 that become sequestered. Here we report that the concentration-dependent capability of DNAJB6 to suppress fibril formation in thioflavin T fluorescence assays decreases progressively with an increasing number of S/T substitutions, with an almost complete loss of suppression when 18 S/T residues are substituted.