10 research outputs found
Zipper-Like Unfolding of Ī²-Sheets Accessed by Pioneer Water Molecules: Atomic Resolution of Forced Unfold Reveals Different Mechanisms for Parallel and Antiparallel Motifs
In this study, quantum mechanical calculations were used for an atomic level investigation of the Ī²-sheet unfolding mechanism aided by pioneer water molecules accessing the structural motif. Results indicate that there is a qualitatively different forced unfold mechanism for parallel and antiparallel Ī²-sheets. In the case of parallel Ī²-sheets, the presence of only a single water molecule could already be enough to stimulate rupture of consecutive backbone hydrogen bonds by stepping from one H-bond to the next one, similarly as a slider opens up a zipper. The extension curves and energetics obtained at the B3LYP/6-311++G(d,p)//B3LYP/6-31G(d) level of theory correlate well and may explain the hyperfine resolution of experimentally observed sawtooth patterns in single molecule studies where external pulling force was applied
Zipper-Like Unfolding of Ī²-Sheets Accessed by Pioneer Water Molecules: Atomic Resolution of Forced Unfold Reveals Different Mechanisms for Parallel and Antiparallel Motifs
In this study, quantum mechanical calculations were used for an atomic level investigation of the Ī²-sheet unfolding mechanism aided by pioneer water molecules accessing the structural motif. Results indicate that there is a qualitatively different forced unfold mechanism for parallel and antiparallel Ī²-sheets. In the case of parallel Ī²-sheets, the presence of only a single water molecule could already be enough to stimulate rupture of consecutive backbone hydrogen bonds by stepping from one H-bond to the next one, similarly as a slider opens up a zipper. The extension curves and energetics obtained at the B3LYP/6-311++G(d,p)//B3LYP/6-31G(d) level of theory correlate well and may explain the hyperfine resolution of experimentally observed sawtooth patterns in single molecule studies where external pulling force was applied
Zipper-Like Unfolding of Ī²-Sheets Accessed by Pioneer Water Molecules: Atomic Resolution of Forced Unfold Reveals Different Mechanisms for Parallel and Antiparallel Motifs
In this study, quantum mechanical calculations were used for an atomic level investigation of the Ī²-sheet unfolding mechanism aided by pioneer water molecules accessing the structural motif. Results indicate that there is a qualitatively different forced unfold mechanism for parallel and antiparallel Ī²-sheets. In the case of parallel Ī²-sheets, the presence of only a single water molecule could already be enough to stimulate rupture of consecutive backbone hydrogen bonds by stepping from one H-bond to the next one, similarly as a slider opens up a zipper. The extension curves and energetics obtained at the B3LYP/6-311++G(d,p)//B3LYP/6-31G(d) level of theory correlate well and may explain the hyperfine resolution of experimentally observed sawtooth patterns in single molecule studies where external pulling force was applied
Hyperfine-Shifted <sup>13</sup>C Resonance Assignments in an IronāSulfur Protein with Quantum Chemical Verification: Aliphatic CāHĀ·Ā·Ā·S 3-Centerā4-Electron Interactions
Although the majority of noncovalent interactions associated with hydrogen and heavy atoms in proteins and other biomolecules are classical hydrogen bonds between polar NāH or OāH moieties and O atoms or aromatic Ļ electrons, high-resolution X-ray crystallographic models deposited in the Protein Data Bank show evidence for weaker CāHĀ·Ā·Ā·O hydrogen bonds, including ones involving sp<sup>3</sup>-hybridized carbon atoms. Little evidence is available in proteins for the (even) weaker CāHĀ·Ā·Ā·S interactions described in the crystallographic literature on small molecules. Here, we report experimental evidence and theoretical verification for the existence of nine aliphatic (sp<sup>3</sup>-hybridized) CāHĀ·Ā·Ā·S 3-centerā4-electron interactions in the protein Clostridium pasteurianum rubredoxin. Our evidence comes from the analysis of carbon-13 NMR chemical shifts assigned to atoms near the iron at the active site of this protein. We detected anomalous chemical shifts for these carbon-13 nuclei and explained their origin in terms of unpaired spin density from the iron atom being delocalized through interactions of the type: CāHĀ·Ā·Ā·SāFe, where S is the sulfur of one of the four cysteine side chains covalently bonded to the iron. These results suggest that polarized sulfur atoms in proteins can engage in multiple weak interactions with surrounding aliphatic groups. We analyze the strength and angular dependence of these interactions and conclude that they may contribute small, but significant, stabilization to the molecule
DYNLL2 Dynein Light Chain Binds to an Extended Linear Motif of Myosin 5a Tail That Has Structural Plasticity
LC8
dynein light chains (DYNLL) are conserved homodimeric eukaryotic
hub proteins that participate in diverse cellular processes. Among
the binding partners of DYNLL2, myosin 5a (myo5a) is a motor protein
involved in cargo transport. Here we provide a profound characterization
of the DYNLL2 binding motif of myo5a in free and DYNLL2-bound form
by using nuclear magnetic resonance spectroscopy, X-ray crystallography,
and molecular dynamics simulations. In the free form, the DYNLL2 binding
region, located in an intrinsically disordered domain of the myo5a
tail, has a nascent helical character. The motif becomes structured
and folds into a Ī²-strand upon binding to DYNLL2. Despite differences
of the myo5a sequence from the consensus binding motif, one peptide
is accommodated in each of the parallel DYNLL2 binding grooves, as
for all other known partners. Interestingly, while the core motif
shows a similar interaction pattern in the binding groove as seen
in other complexes, the flanking residues make several additional
contacts, thereby lengthening the binding motif. The N-terminal extension
folds back and partially blocks the free edge of the Ī²-sheet
formed by the binding motif itself. The C-terminal extension contacts
the dimer interface and interacts with symmetry-related residues of
the second myo5a peptide. The involvement of flanking residues of
the core binding site of myo5a could modify the quaternary structure
of the full-length myo5a and affect its biological functions. Our
results deepen the knowledge of the diverse partner recognition of
DYNLL proteins and provide an example of a Janus-faced linear motif
Phosphorylation as Conformational Switch from the Native to Amyloid State: Trp-Cage as a Protein Aggregation Model
The 20 residue long Trp-cage miniprotein
is an excellent model
for both computational and experimental studies of protein folding
and stability. Recently, great attention emerged to study disease-related
protein misfolding, aggregation, and amyloid formation, with the aim
of revealing their structural and thermodynamic background. Trp-cage
is sensitive to both environmental and structure-modifying effects.
It aggregates with ease upon structure destabilization, and thus it
is suitable for modeling aggregation and amyloid formation. Here,
we characterize the amyloid formation of several sequence modified
and side-chain phosphorylated Trp-cage variants. We applied NMR, circular
dichroism (CD) and Fourier transform infrared (FTIR) spectroscopies,
molecular dynamics (MD) simulations, and transmission electron microscopy
(TEM) in conjunction with thioflavin-T (ThT) fluorescence measurements
to reveal the structural consequences of side-chain phosphorylation.
We demonstrate that the native fold is destabilized upon serine phosphorylation,
and the resultant highly dynamic structures form amyloid-like ordered
aggregates with high intermolecular Ī²-structure content. The
only exception is the D9SĀ(P) variant, which follows an alternative
aggregation process by forming thin fibrils, presenting a CD spectrum
of PPII helix, and showing low ThT binding capability. We propose
a complex aggregation model for these Trp-cage miniproteins. This
model assumes an additional aggregated state, a collagen triple helical
form that can precede amyloid formation. The phosphorylation of a
single serine residue serves as a conformational switch, triggering
aggregation, otherwise mediated by kinases in cell. We show that Trp-cage
miniprotein is indeed a realistic model of larger globular systems
of composite folding and aggregation landscapes and helps us to understand
the fundamentals of deleterious protein aggregation and amyloid formation
DYNLL2 Dynein Light Chain Binds to an Extended Linear Motif of Myosin 5a Tail That Has Structural Plasticity
LC8
dynein light chains (DYNLL) are conserved homodimeric eukaryotic
hub proteins that participate in diverse cellular processes. Among
the binding partners of DYNLL2, myosin 5a (myo5a) is a motor protein
involved in cargo transport. Here we provide a profound characterization
of the DYNLL2 binding motif of myo5a in free and DYNLL2-bound form
by using nuclear magnetic resonance spectroscopy, X-ray crystallography,
and molecular dynamics simulations. In the free form, the DYNLL2 binding
region, located in an intrinsically disordered domain of the myo5a
tail, has a nascent helical character. The motif becomes structured
and folds into a Ī²-strand upon binding to DYNLL2. Despite differences
of the myo5a sequence from the consensus binding motif, one peptide
is accommodated in each of the parallel DYNLL2 binding grooves, as
for all other known partners. Interestingly, while the core motif
shows a similar interaction pattern in the binding groove as seen
in other complexes, the flanking residues make several additional
contacts, thereby lengthening the binding motif. The N-terminal extension
folds back and partially blocks the free edge of the Ī²-sheet
formed by the binding motif itself. The C-terminal extension contacts
the dimer interface and interacts with symmetry-related residues of
the second myo5a peptide. The involvement of flanking residues of
the core binding site of myo5a could modify the quaternary structure
of the full-length myo5a and affect its biological functions. Our
results deepen the knowledge of the diverse partner recognition of
DYNLL proteins and provide an example of a Janus-faced linear motif
Rational Design of Ī±āHelix-Stabilized Exendinā4 Analogues
Exendin-4 (Ex4) is a potent glucagon-like
peptide-1 receptor agonist,
a drug regulating the plasma glucose level of patients suffering from
type 2 diabetes. The moleculeās poor solubility and its readiness
to form aggregates increase the likelihood of unwanted side effects.
Therefore, we designed Ex4 analogues with improved structural characteristics
and better water solubility. Rational design was started from the
parent 20-amino acid, well-folded Trp cage (TC) miniprotein and involved
the step-by-step N-terminal elongation of the TC head, resulting in
the 39-amino acid Ex4 analogue, E19. Helical propensity coupled to
tertiary structure compactness was monitored and quantitatively analyzed
by electronic circular dichroism and nuclear magnetic resonance (NMR)
spectroscopy for the 14 peptides of different lengths. Both <sup>15</sup>N relaxation- and diffusion-ordered NMR measurements were established
to investigate the inherent mobility and self-association propensity
of Ex4 and E19. Our designed E19 molecule has the same tertiary structure
as Ex4 but is more helical than Ex4 under all studied conditions;
it is less prone to oligomerization and has preserved biological activity.
These conditions make E19 a perfect lead compound for further drug
discovery. We believe that this structural study improves our understanding
of the relationship between local molecular features and global physicochemical
properties such as water solubility and could help in the development
of more potent Ex4 analogues with improved pharmacokinetic properties
Atropisomerism of the Asn Ī± Radicals Revealed by Ramachandran Surface Topology
C radicals are typically trigonal
planar and thus achiral, regardless
of whether they originate from a chiral or an achiral C-atom (e.g.,
CāH + <sup>ā¢</sup>OH ā Cā¢ + H<sub>2</sub>O). <b>Oxidative stress</b> could initiate radical formation
in proteins when, for example, the H-atom is abstracted from the CĪ±-carbon
of an amino acid residue. Electronic structure calculations show that
such a radical remains achiral when formed from the achiral Gly, or
the chiral but small Ala residues. However, when longer side-chain
containing proteogenic amino acid residues are studied (e.g., Asn),
they provide radicals of axis chirality, which in turn leads to <b>atropisomerism</b> observed for the first time for peptides. The
two <b>enantiomeric</b> extended backbone <b>structures</b>, ā¢Ī²<sub>L</sub> and ā¢Ī²<sub>D</sub>, interconvert
via a pair of <b>enantiotopic reaction paths</b>, monitored
on a 4D Ramachandran surface, with two distinct transition states
of very different <i>Gibbs</i>-free energies: 37.4 and 67.7
kJ/mol, respectively. This discovery requires the reassessment of
our understanding on radical formation and their conformational and
stereochemical behavior. Furthermore, the atropisomerism of proteogenic
amino acid residues should affect our understanding on radicals in
biological systems and, thus, reframes the role of the D-residues
as markers of <b>molecular aging</b>
Secondary Structure of Short Ī²āPeptides as the Chiral Expression of Monomeric Building Units: A Rational and Predictive Model
Chirality of the monomeric residues controls and determines
the
prevalent folding of small oligopeptides (from di- to tetramers) composed
of 2-aminocyclobutane-1-carboxylic acid (ACBA) derivatives with the
same or different absolute and relative configuration. The <i>cis</i>-form of the monomeric ACBA gives rise to two conformers,
namely, Z6 and Z8, while the <i>trans</i>-form manifests
uniquely as an H8 structure. By combining these subunits in oligo-
and polypeptides, their local structural preference remains, thus
allowing the rational design of new short foldamers. A lego-type molecular
architecture evolves; the overall look depends only on the conformational
properties of the structural building units. A versatile and efficient
method to predict the backbone folds of designed cyclobutane Ī²-peptides
is based on QM calculations. Predictions are corroborated by high-resolution
NMR studies on selected stereoisomers, most of them being new foldamers
that have been synthesized and characterized for the first time. Thus,
the chiral expression of monomeric building units results in the defined
secondary structures of small oligomers. As a result of this study,
a new set of chirality controlled foldamers is provided to probe as
biocompatible biopolymers