Methyl group dynamics as a probe of the protein dynamical transition

Hydrated proteins undergo a dynamical transition around 200 K from glasslike to liquidlike motion. Molecular dynamics simulations have been used to study the temperature dependence of the dynamics of ribonuclease A in the hydrated crystal, a model dehydrated powder, and aqueous solution. Changes in the dynamics accompanying the transition throughout the protein have been quantified in terms of the mean-squared fluctuations (MSFs) of methyl hydrogen atoms on the 100 ps time scale. In solution at 300 K the MSFs span a broad distribution, consistent with NMR relaxation measurements. The MSF distribution in the hydrated crystal at 300 K is qualitatively similar to the solution result, except for a slight shift to lower values, and dehydration results in a dramatic shift of the MSFs to lower values. As the temperature is lowered, the whole distribution of methyl group fluctuations in the hydrated crystal shifts to lower values. Most of the methyl groups in the hydrated protein display a nonlinear temperature dependence with a dynamical transition at approximately 200 K, but most methyl groups do not undergo a transition in the dehydrated protein. We conclude that the dynamical transition occurs throughout most of the protein and that solvent is required for the transition.     

Alanine methyl groups as NMR probes of molecular structure and dynamics in high-molecular-weight proteins

The importance and utility of Ala(β) methyl groups as NMR probes of molecular structure and dynamics in high-molecular-weight proteins is explored. Using (2)H and (13)C relaxation measurements in {U-(2)H; Ala(β)-[(13)CHD(2)]}-labeled Malate Synthase G (MSG)--an 82-kDa monomeric enzyme that contains 73 Ala(β) methyl groups--we show that the vast majority of selectively labeled Ala(β) methyls are highly ordered. A number of NMR applications used for solution studies of structure and dynamics of large protein molecules can benefit from proximity of Ala(β) methyls to the protein backbone and their high degree of ordering. In the case of MSG, these applications include the measurement of (1)H-(13)C residual dipolar couplings in Ala(β) methyls, characterization of slow (μs-to-ms) dynamics at the substrates' binding sites, and methyl-TROSY-based NOE spectroscopy performed on {U-(2)H; Ala(β)-[(13)CH(3)]; Ile(δ1)-[(13)CH(3)]; Leu,Val-[(13)CH(3)/(12)CD(3)]}-labeled samples where the number of methyl probes for derivation of distance restraints is maximized compared to the state-of-the-art ILV labeling methodology.     

Site-specific incorporation of a (19)F-amino acid into proteins as an NMR probe for characterizing protein structure and reactivity

19F NMR is a powerful tool for monitoring protein conformational changes and interactions; however, the inability to site-specifically introduce fluorine labels into proteins of biological interest severely limits its applicability. Using methods for genetically directing incorporation of unnatural amino acids, we have inserted trifluoromethyl-l-phenylalanine (tfm-Phe) into proteins in vivo at TAG nonsense codons with high translational efficiency and fidelity. The binding of substrates, inhibitors, and cofactors, as well as reactions in enzymes, were studied by selective introduction of tfm-Phe and subsequent monitoring of the 19F NMR chemical shifts. Subtle protein conformational changes were detected near the active site and at long distances (25 Angstrom). 19F signal sensitivity and resolution was also sufficient to differentiate protein environments in vivo. Since there has been interest in using 19F-labeled proteins in solid-state membrane protein studies, folding studies, and in vivo studies, this general method for genetically incorporating a 19F-label into proteins of any size in Escherichia coli should have broad application beyond that of monitoring protein conformational changes.     

Paramagnetic cobalt(II) as an NMR probe of dendrimer structure: mobility and cooperativity of dendritic arms

Cobalt(II) has been utilized as an external paramagnetic (1)H NMR probe for the study of the structure of dendrimers that possess specifically located metal recognition sites. The hyperfine-shifted (1)H NMR signals of the Co(II) complexes of several 2,6-diamidopyridine-containing dendrimers have been fully assigned by means of 1D and 2D NMR techniques, including NOE difference, EXSY, COSY, and TOCSY. Temperature-dependent T(1) values of the hyperfine-shifted signals were used to conclude that the Co(II)-dendrimer complexes are in the "liquidlike" regime, indicative of a shell-like structure instead of a "dense-core" structure. The presence of sizable cavities within the dendrimers was observed including a loosely packed conformation for the 2,6-diamidopyridino moiety to bind to potential guest molecules. Cooperativity among the dendritic arms in metal binding is also observed, whereby two dendritic arms bind to the metal center at the same time. In the case of dendrimers with the metal binding site located near the surface of the molecule, such binding cooperativity is still observed despite the large degree of freedom of the metal-binding moiety. Cooperativity among the dendritic arms can thus be considered an intrinsic property, which has to be taken into consideration in future design of functional dendrimers for the purpose of specific recognition and catalysis. The hydrodynamic radii of these dendrimers have been determined by means of nuclear Overhouser effect at low temperature. The study offers a method for the study of the dynamics of dendrimers in solution under different conditions and upon ligand binding and recognition. The study also provides a tool for monitoring systematic variation of the metal binding site in different dendrimer frameworks for specific applications, such as catalysis and molecular recognition.     

Simultaneous NMR study of protein structure and dynamics using conservative mutagenesis

A novel iterative procedure is described that allows both the orientation and dynamics of internuclear bond vectors to be determined from direct interpretation of NMR dipolar couplings, measured under at least three orthogonal alignment conditions. If five orthogonal alignments are available, the approach also yields information on the degree of motional anisotropy and the direction in which the largest amplitude internal motion of each bond vector takes place. The method is demonstrated for the backbone (15)N-(1)H, (13)C(alpha)-(1)H(alpha), and (13)C(alpha)-13C' interactions in the previously well-studied protein domain GB3, dissolved in a liquid crystalline suspension of filamentous phage Pf1. Alignment variation is achieved by using conservative mutations of charged surface residues. Results indicate remarkably uniform backbone dynamics, with amplitudes that agree well with those of previous (15)N relaxation studies for most residues involved in elements of secondary structure, but larger amplitude dynamics than those found by (15)N relaxation for residues in loop and turn regions. In agreement with a previous analysis of dipolar couplings, the N-H bonds in the second beta-strand, which is involved in antibody recognition, show elevated dynamics with largest amplitudes orthogonal to the chain direction.     

Anesthetic modulation of protein dynamics: insight from an NMR study

Mistic (membrane integrating sequence for translation of integral membrane protein constructs) comprises the four-alpha-helix bundle scaffold found in the transmembrane domains of the Cys-loop receptors that are plausible targets for general anesthetics. Nuclear magnetic resonance (NMR) studies of anesthetic halothane interaction with Mistic in dodecyl phosphocholine (DPC) micelles provide an experimental basis for understanding molecular mechanisms of general anesthesia. Halothane was found to interact directly with Mistic, mostly in the interfacial loop regions. Although the presence of halothane had little effect on Mistic structure, (15)N NMR relaxation dispersion measurements revealed that halothane affected Mistic's motion on the microsecond-millisecond time scale. Halothane shifted the equilibrium of chemical exchange in some residues and made the exchange faster or slower in comparison to the original state in the absence of halothane. The motion on the microsecond-millisecond time scale in several residues disappeared in response to the addition of halothane. Most of the residues experiencing halothane-induced dynamics changes also exhibited profound halothane-induced changes in chemical shift, suggesting that dynamics modification of these residues might result from their direct interaction with halothane molecules. Allosteric modulation by halothane also contributed to dynamics changes, as reflected in residues I52 and Y82 where halothane introduction brought about dynamics changes but not chemical shift changes. The study suggests that inhaled general anesthetics could act on proteins via altering protein motion on the microsecond-millisecond time scale, especially motion in the flexible loops that link different alpha helices. The validation of anesthetic effect on protein dynamics that are potentially correlated with protein functions is a critical step in unraveling the mechanisms of anesthetic action on proteins.     

Toward the supramolecular structure of collagen: a molecular dynamics approach

The structure, stability, and conformational dynamics of an assembly of two pentameric bundles made of collagen-like triple helical segments are explored using 1.2 ns molecular dynamics simulations in three environments: 8.0% (v/v) formaldehyde/water solution, 1.4% (v/v) gallic acid/water solution, and pure water. Stable supramolecular arrangements, where the two collagen units are very close to each other at interacting distances, are identified via docking and energy minimization procedures. Analysis of the interaction with formaldehyde and gallic acid suggests that they perturb the protein in a similar way depending on hydrogen-bonding capability, hydrophobic association properties, and the size and concentration of the compound.     

Temporal gradients in microfluidic systems to probe cellular dynamics: A review

Microfluidic devices have found a unique place in cellular studies due to the ease of fabrication, their ability to provide long-term culture, or the seamless integration of downstream measurements into the devices. The accurate and precise control of fluid flows also allows unique stimulant profiles to be applied to cells that have been difficult to perform with conventional devices. In this review, we describe and provide examples of microfluidic systems that have been used to generate temporal gradients of stimulants, such as waveforms or pulses, and how these profiles have been used to produce biological insights into mammalian cells that are not typically revealed under static concentration gradients. We also discuss the inherent analytical challenges associated with producing and maintaining temporal gradients in these devices.     

Laser light scattering as a probe for structure and dynamics in bulk polymers: Polystyrene

Summary We begin with an outline of a theory of density fluctuations in the glassy state based on a partial freeze-in of an ordering parameter, as derived from previous equation of state investigations. Then laser light scattering studies of glasses formed by atactic polystyrene are described. Photon correlation analysis indicates two relaxation mechanisms. A slow process dominates aboveT _g , has a wide asymmetric distribution of relaxation times and is evidently related to the glass transition phenomenon. A small-amplitude fast decay with a narrow relaxation spectrum is also observed. BelowT _g , this mode is shown to be dependent on scattering angle and is evidently a diffusion mechanism, which may be related to the-relaxation process. Relative integrated intensities are presented for isotropic and depolarized scattering from glasses densified by pressurization in the melt, followed by isobaric cooling to 25°C and depressurization. The depolarized scattering for the glass formed at atmospheric pressure has a value consistent with a completely disordered glass. However, a small increase in depolarized scattering appears to occur on densification. This result may reflect a small increase in intersegmental ordering in glassy polystyrene upon densification. The isotropic scattering shows a large systematic increase in the densified glass. This is apparently the result of an inhomogeneous density change during the depressurization step.

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Zusammenfassung Wir beginnen mit dem Umriß einer allgemeinen Theorie der Dichteschwankungen im glasigen Bereich. Sie beruht auf der Annahme despartiellen Einfrierens eines Struktur-Parameters, wie aus vorherigen Untersuchungen der Zustandsgleichung gefolgert wird. Sodann werden Versuche mit ataktischem Polystyrol beschrieben. Die zeitliche Korrelationsfunktion der Photonenstreuung zeigt zwei Relaxationsmechanismen an. OberhalbT _g herrscht ein langsamer Prozeß vor, der durch eine breite, assymetrische Verteilungsfunktion von Relaxationszeiten charakterisiert ist. Dieser Prozeß hängt offensichtlich mit dem Glasübergang zusammen. Ein zusätzlicher, viel rascherer Prozeß mit einer geringen Amplitude und einem schmalen Relaxationsspektrum ist auch beobachtbar. UnterhalbT _g wird dieser Prozeß vom Streuwinkel abhängig, stellt einen Diffusionsvorgang dar, und sollte mit dem ß-Prozeß zusammenhängen. Weiter untersuchen wir die relativen totalen Intensitäten der isotropen und der depolarisierten Streuungskomponente für eine Reihe von verdichteten Gläsern. Diese wurden durch Druckanwendung in der Schmelze, nachherige isobarische Abkühlung zu 25 °C, und schließliche Druckentlastung erzeugt. Die depolarisierte Streuungskomponente im Glase, welches bei atmosphärischem Druck hergestellt wurde, ist mit einem System ohne sichtliche Ordnungszustände vereinbar. Ein schwacher Anstieg dieser Streuung wird jedoch im verdichteten System bemerkbar, welcher möglicherweise einen geringeren Ordnungsprozeß der Kettensegmente anzeigt. Die isotropische Streuung wächst systematisch mit der glasigen Verdichtung an. Offensichtlich ist dies die Folge einer inhomogenen Dichteänderung während der Druckentlastung.

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Dedicated to Professor Dr. G. Rehage on the occasion of his 60th birthday.

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With 7 figures and 1 table     

Determination of membrane protein structure and dynamics by magic-angle-spinning solid-state NMR spectroscopy

It is shown that molecular structure and dynamics of a uniformly labeled membrane protein can be studied under magic-angle-spinning conditions. For this purpose, dipolar recoupling experiments are combined with novel through-bond correlation schemes that probe mobile protein segments. These NMR schemes are demonstrated on a uniformly [13C,15N] variant of the 52-residue polypeptide phospholamban. When reconstituted in lipid bilayers, the NMR data are consistent with an alpha-helical trans-membrane segment and a cytoplasmic domain that exhibits a high degree of structural disorder.     

Nuclear spin conversion to probe the methyl rotation effect on hydrogen-bond and vibrational dynamics

A noteworthy example of a molecule with coupled large-amplitude motions is provided by acetylacetone (methyl group torsions and intramolecular hydrogen bonds). The molecule was trapped in solid parahydrogen to investigate the complex proton tunneling processes. Nuclear spin conversion in methyl groups is observed and, combined with IR spectra, documents the coupling between high frequency modes and large amplitude motions.     

Oxygen as a paramagnetic probe of membrane protein structure by cysteine mutagenesis and (19)F NMR spectroscopy

Oxygen solubility increases toward the hydrophobic interior of membranes. Using NMR, this O(2) solubility gradient gives rise to an exquisite range of position-dependent paramagnetic effects at partial pressures of 100 atm (PO(2)), which may be used to probe membrane protein structure and positioning. In this study, fluorinated probes were introduced at selected positions of the transmembrane 1 domain of the intact homotrimer of the integral membrane protein, diacylglycerol kinase. Using (19)F NMR, O(2)-induced chemical shift perturbations revealed secondary structure, membrane immersion depth, and regions of the helix in contact with the protein or with the micelle.     

Fluorescent probe as reporter on the local structure and dynamics in hydrolysis-condensation process of organotrialkoxysilanes

To get some information on the aggregation behaviors of the products derived from different organotrialkoxysilanes, the hydrolysis-condensation processes of some organotrialkoxysilanes have been examined by means of pyrene as fluorescent probe. The organotrialkoxysilanes used in the research were n-octadecyltri-methoxysilane (ODTMS), n-octyltrimethoxysilane (OTMS), 3-glycidoxypropyltrimethoxysilane (GTMS), 3-methacryloxypropyltrimethoxysilane (MAPTMS), and propyltrimethoxy-silane (PTMS). The results show that pyrene as fluorescence probe can respond sensitively not only to the organization state of the hydrolysates but also to the change in the organization state during the condensation process. The organization states during the hydrolysis and condensation can be explained in terms of structures of the products. In the initial stage, the silanols with long organic chains are amphiphilic molecules, and such nature of the silanols can be compared to that of a surfactant. Therefore, the excimer emission of pyrene is extremely obvious because of such silanols being prone to form aggregates. In the case of silanols having short alkyl groups or epoxy groups, these silanols homogenously disperse in solution, which results in the appearance of an only monomer emission of pyrene. In the late stage, the fluorescence behavior of pyrene is also sensitive to structural evolution of the silicates. The fluorescence spectra of pyrene during the condensation of the silanols with short alkyl groups or epoxy groups are almost in silence, indicating that the condensation products, with a low condensation degree, homogeneously disperse in solution. For the silanols with long hydrophobic substituents in different lengths, the changes in fluorescence spectra of pyrene during the condensation are varied. Commonly, the excimer emission is noticeable, implying that the condensation products with high condensation degree inhomogenously disperse in solution. However, the relative excimer/monomer fluorescence intensity is alkyl chain-length dependent. The longer alkyl chains in the condensation products result in the appearance of the obvious excimer emission. These phenomena imply that the condensation degree of the products increases with the length of the alkyl chains. Additionally, the distorted spectrum of pyrene appears in the case of the organotrialkoxysilanes with side chain substituent, illustrating that the steric hindrance between the substituents can be monitored by fluorescence of pyrene. All these results are verified by the fluorescence-quenching measurements. The approach in the present study gives new insights into the local structure and dynamics in hydrolysis-condensation process of organotrialkoxysilanes and emphasizes the influence of the self-assembling behavior.     

Activators of cylindrical proteases as antimicrobials: identification and development of small molecule activators of ClpP protease

ClpP is a cylindrical serine protease whose ability to degrade proteins is regulated by the unfoldase ATP-dependent chaperones. ClpP on its own can only degrade small peptides. Here, we used ClpP as a target in a high-throughput screen for compounds, which activate the protease and allow it to degrade larger proteins, hence, abolishing the specificity arising from the ATP-dependent chaperones. Our screen resulted in five distinct compounds, which we designate as Activators of Self-Compartmentalizing Proteases 1 to 5 (ACP1 to 5). The compounds are found to stabilize the ClpP double-ring structure. The ACP1 chemical structure was considered to have drug-like characteristics and was further optimized to give analogs with bactericidal activity. Hence, the ACPs represent classes of compounds that can activate ClpP and that can be developed as potential novel antibiotics.     

Real-time NMR characterization of structure and dynamics in a transiently populated protein folding intermediate

Recent advances in NMR spectroscopy and the availability of high magnetic field strengths now offer the possibility to record real-time 3D NMR spectra of short-lived protein states, e.g., states that become transiently populated during protein folding. Here we present a strategy for obtaining sequential NMR assignments as well as atom-resolved information on structural and dynamic features within a folding intermediate of the amyloidogenic protein β2-microglobulin that has a half-lifetime of only 20 min.     

Structure,dynamics and interactions in polymer systems as studied by NMR spectroscopy

The possibilities of NMR spectroscopy in studies of interactions in polymer systems are demonstrated on the example of two types of macromolecular complexes: (i) By measuring ¹H NMR high resolution line intensities, the formation of ordered associated structures of syndiotactic (s) poly(methyl methacrylate)(PMMA) in mixed solvents was quantitatively characterized. The obtained results permit us to assume that the mechanism by which the solvent affects self-association of s-PMMA involves specific interactions of the solvent molecules with PMMA units. Solid state high resolution ¹³C NMR spectra of associated s-PMMA gels were also measured and compared with the spectra of a solid s-PMMA sample. (ii) By using ¹³C solid state NMR spectroscopy, the differences in the structure of the amorphous and crystalline phases in pure poly(ethylene oxide) and its complexes with p-dichlorobenzene or p-nitrophenol were characterized. Prounounced differences also in the dynamic structure of the crystalline phase in these systems are indicated by the relaxation times T₁(C), T_1ρ(C) and T_1ρ(H).     

Behavior of liquid crystals confined to mesoporous materials as studied by 13C NMR spectroscopy of methyl iodide and methane as probe molecules

The behavior of thermotropic nematic liquid crystals (LCs) Merck Phase 4 and ZLI 1115 confined to mesoporous controlled pore glass materials was investigated using 13C nuclear magnetic resonance spectroscopy of probe molecules methyl iodide and methane. The average pore diameters of the materials varied from 81 to 375 A, and the temperature series measurements were performed on solid, nematic, and isotropic phases of bulk LCs. Chemical shift, intensity, and line shape of the resonance signals in the spectra contain lots of information about the effect of confinement on the state of the LCs. The line shape of the 13C resonances of the CH3I molecules in LCs confined into the pores was observed to be even more sensitive to the LC orientation distribution than, for example, that of 2H spectra of deuterated LCs or 129Xe spectra of dissolved xenon gas. The effect of the magnetic field on the orientation of LC molecules inside the pores was examined in four different magnetic fields varying from 4.70 to 11.74 T. The magnetic field was found to have significant effect on the orientation of LC molecules in the largest pores and close to the nematic-isotropic phase transition temperature. The theoretical model of shielding of noble gases dissolved in LCs based on pairwise additivity approximation was utilized in the analysis of CH4 spectra. For the first time, a first-order nematic-isotropic phase transition was detected to take place inside such restrictive hosts. In the larger pores a few degrees below the nematic-isotropic phase transition of bulk LC the 13C quartet of CH3I changes as a powder pattern. Results are compared to those derived from 129Xe NMR measurements of xenon gas in similar environments.     

NMR studies of the dynamics of a bifunctional rhodamine probe attached to troponin C

Fluorescence polarization measurements of bifunctional rhodamine (BR) probes provide a powerful approach to determine the in situ orientation of proteins within ordered complexes such as muscle fibers. For accurate interpretation of fluorescence measurements, it is important to understand the probe dynamics relative to the protein to which it is attached. We previously determined the structure of the N-domain of chicken skeletal troponin C, BR-labeled on the C helix, in complex with the switch region of troponin I, and demonstrated that the probe does not perturb the structure or dynamics of the protein. In this study, the motion of the fluorescence label relative to the protein has been characterized using NMR relaxation measurements of 13C-labeled methyl groups on the BR probe and 15N-labeled backbone amides of the protein. Probe dynamics were monitored using off-resonance 13C-R(1rho), 13C-R(1) and {1H}-13C NOE at magnetic field strengths of 500, 600, and 800 MHz. Relaxation data were interpreted in terms of the overall rotational correlation time of the protein and a two-time scale model for internal motion of the BR methyl groups, using a numerical optimization with Monte Carlo parameter error estimation. The analysis yields a 1.5 +/- 0.4 ps correlation time for rotation around the three-fold methyl symmetry axis, and a 0.8 +/- 0.4 ns rotational correlation time for reorientation of the 13C-14N bond with an associated S2s of 0.79 +/- 0.03. Order parameters of the backbone NH vectors in the helix to which the probe is attached average S2 approximately 0.85, implying that the amplitude of independent reorientation of the BR probe is small in magnitude, consistent with results from fluorescence polarization measurements in reconstituted muscle fibers.