Effective Rotational Correlation Time Research Articles

Accurate Measurement of Residual Dipolar Couplings in Large RNAs by Variable Flip Angle NMR.

NMR approaches using nucleotide-specific deuterium labeling schemes have enabled structural studies of biologically relevant RNAs of increasing size and complexity. Although local structure is well-determined using these methods, definition of global structural features, including relative orientations of independent helices, remains a challenge. Residual dipolar couplings, a potential source of orientation information, have not been obtainable for large RNAs due to poor sensitivity resulting from rapid heteronuclear signal decay. Here we report a novel multiple quantum NMR method for RDC determination that employs flip angle variation rather than a coupling evolution period. The accuracy of the method and its utility for establishing interhelical orientations are demonstrated for a 36-nucleotide RNA, for which comparative data could be obtained. Applied to a 78 kDa Rev response element from the HIV-1 virus, which has an effective rotational correlation time of ca. 160 ns, the method yields sensitivity gains of an order of magnitude or greater over existing approaches. Solution-state access to structural organization in RNAs of at least 230 nucleotides is now possible.

EPR SPECTROSCOPY STUDIES OF CHANGES IN ERYTHROCYTE MEMBRANES IN PATIENTS WITH LARYNGEAL CANCER

To evaluate microviscosity and sorption capacity of erythrocyte membranes (SCEM) from patients with laryngeal cancer (LC). Samples from 35 patients with LC of stages II and III and 20 healthy volunteers were investigated by electron paramagnetic resonance with Bis(1-oxyl-2,2,6,6-tetramethylpiperidinyl-4)-ester of 5,7-dimethyladamantane-1,3-dicarbonic acid (AdTEMPO) probe. SCEM was evaluated by amount of unabsorbed methylene blue. Microviscosity of erythrocyte membranes was determined by the effective rotational diffusion correlation times (τeff) and a decrease in radical spectrum signal intensity per hour. The most apparent decrease in mobility of the AdTEMPO in erythrocytes was observed prior to washing of erythrocytes with 0.9% NaCl for 5 min after probe insertion. The deceleration after 60 min was observed only in stage II LC. τeff was at control values after washing of erythrocytes of stage II LC 5 min after probe insertion and was significantly reduced in stage III LC in comparison to control. Radical spectrum signal intensity per hour in samples of stage II and III patients prior to and after washing of erythrocytes was on average 1.5-fold higher than that of control. SCEM in samples of stage II and III LC was found in 40 and 33% cases, respectively and was on average significantly reduced in comparison to control. The initial interaction of AdTEMPO with erythrocyte membranes of stage II and III LC patients is accompanied by an increase in τeff, indicating deceleration of probe rotation. τeff of the probe in membranes remains unchanged in 60 min, indicating changes in the structural organization of lipid bilayer and its associated proteins in particular. The similarity of SCEM for both studied groups reflects the pathological changes in function of erythrocyte membranes.

HYCUD: a computational tool for prediction of effective rotational correlation time in flexible proteins.

A large fraction of eukaryotic proteins contain unstructured tails or linkers. The presence of flexible regions allows these systems to experience a high level of mobility facilitating their biological function. The complex nature of protein rotation in such flexible modular systems precludes a straightforward application of hydrodynamic methods to calculate their rotational motional properties. We describe the workflow of HYdrodynamic CoUpling of Domains (HYCUD), a program for prediction of effective rotational correlation times in multidomain proteins. The usage of HYCUD is demonstrated by its application to the ribosomal protein L7/L12. Rotational correlation times predicted by HYCUD might be used to detect molecular switch events mediated by disorder-order transitions in interdomain linkers. The source code and documentation are available at www.mpibpc.mpg.de/106144/software. mzwecks@gwdg.de or nare@nmr.mpibpc.mpg.de Supplementary material is available at Bioinformatics online.

Translational, rotational and internal dynamics of amyloid β-peptides (Aβ40 and Aβ42) from molecular dynamics simulations

In this study, diffusion constants [translational (D(T)) and rotational (D(R))], correlation times [rotational (tau(rot)) and internal (tau(int))], and the intramolecular order parameters (S(2)) of the Alzheimer amyloid-beta peptides Abeta40 and Abeta42 have been calculated from 150 ns molecular dynamics simulations in aqueous solution. The computed parameters have been compared with the experimentally measured values. The calculated D(T) of 1.61 x 10(-6) cm(2)/s and 1.43 x 10(-6) cm(2)/s for Abeta40 and Abeta42, respectively, at 300 K was found to follow the correct trend defined by the Debye-Stokes-Einstein relation that its value should decrease with the increase in the molecular weight. The estimated D(R) for Abeta40 and Abeta42 at 300 K are 0.085 and 0.071 ns(-1), respectively. The rotational (C(rot)(t)) and internal (C(int)(t)) correlation functions of Abeta40 and Abeta42 were observed to decay at nano- and picosecond time scales, respectively. The significantly different time decays of these functions validate the factorization of the total correlation function (C(tot)(t)) of Abeta peptides into C(rot)(t) and C(int)(t). At both short and long time scales, the Clore-Szabo model that was used as C(int)(t) provided the best behavior of C(tot)(t) for both Abeta40 and Abeta42. In addition, an effective rotational correlation time of Abeta40 is also computed at 18 degrees C and the computed value (2.30 ns) is in close agreement with the experimental value of 2.45 ns. The computed S(2) parameters for the central hydrophobic core, the loop region, and C-terminal domains of Abeta40 and Abeta42 are in accord with the previous studies.

Lipid-protein nanodiscs: Possible application in high-resolution NMR investigations of membrane proteins and membrane-active peptides

High-resolution NMR is shown to be applicable for investigation of membrane proteins and membrane-active peptides embedded into lipid-protein nanodiscs (LPNs). (15)N-Labeled K+-channel from Streptomyces lividans (KcsA) and the antibiotic antiamoebin I from Emericellopsis minima (Aam-I) were embedded in LPNs of different lipid composition. Formation of stable complexes undergoing isotropic motion in solution was confirmed by size-exclusion chromatography and (31)P-NMR spectroscopy. The 2D 1H-(15)N-correlation spectra were recorded for KcsA in the complex with LPN containing DMPC and for Aam-I in LPNs based on DOPG, DLPC, DMPC, and POPC. The spectra recorded were compared with those in detergent-containing micelles and small bicelles commonly used in high-resolution NMR spectroscopy of membrane proteins. The spectra recorded in LPN environments demonstrated similar signal dispersion but significantly increased (1)H(N) line width. The spectra of Aam-I embedded in LPNs containing phosphatidylcholine showed significant selective line broadening, thus suggesting exchange process(es) between several membrane-bound states of the peptide. (15)N relaxation rates were measured to obtain the effective rotational correlation time of the Aam-I molecule. The obtained value (approximately 40 nsec at 45 degrees C) is indicative of additional peptide motions within the Aam-I/LPN complex.

Protein effective rotational correlation times from translational self-diffusion coefficients measured by PFG-NMR

Molecular rotational correlation times are of interest for many studies carried out in solution, including characterization of biomolecular structure and interactions. Here we have evaluated the estimates of protein effective rotational correlation times from their translational self-diffusion coefficients measured by pulsed-field gradient NMR against correlation times determined from both collective and residue-specific 15N relaxation analyses and those derived from 3D structure-based hydrodynamic calculations. The results show that, provided the protein diffusive behavior is coherent with the Debye–Stokes–Einstein model, translational diffusion coefficients provide rapid estimates with reasonable accuracy of their effective rotational correlation times. Effective rotational correlation times estimated from translational diffusion coefficients may be particularly beneficial in cases where i) isotopically labelled material is not available, ii) collective backbone 15N relaxation rates are difficult to interpret because of the presence of flexible termini or loops, or iii) a full relaxation analysis is practically difficult because of limited sensitivity owing to low protein concentration, high molecular mass or low temperatures.

Sequence-Specific Resonance Assignment of Soluble Nonglobular Proteins by 7D APSY-NMR Spectroscopy

Based on sequence-specific resonance assignments, NMR is the method of choice for obtaining atomic-resolution experimental data on soluble nonglobular proteins. So far, however, NMR assignment of unfolded polypeptides in solution has been a time-consuming task, mainly due to the small chemical shift dispersion, which has limited practical applications of the NMR approach. This paper presents an efficient, fully automated method for sequence-specific backbone and beta-carbon NMR assignment of soluble nonglobular proteins with sizes up to at least 150 residues. The procedure is based on new APSY (automated projection spectroscopy) experiments which benefit from the short effective rotational correlation times in soluble nonglobular polypeptides to record five- to seven-dimensional NMR data sets, which reliably resolves chemical shift degeneracies. Fully automated sequence-specific resonance assignments of the backbone nuclei and C(beta) are described for the uniformly (13)C,(15)N-labeled urea-denatured 148-residue outer membrane protein X (OmpX) from E. coli. The method is generally applicable to systems with similar spectroscopic properties as unfolded OmpX, and we anticipate that this paper may open the door for extensive atomic-resolution studies of chemical denaturant-unfolded proteins, as well as some classes of functional nonglobular polypeptides in solution.

Regulatory and catalytic domain dynamics of smooth muscle myosin filaments.

Domain dynamics of the chicken gizzard smooth muscle myosin catalytic domain (heavy chain Cys-717) and regulatory domain (regulatory light chain Cys-108) were determined in the absence of nucleotides using saturation-transfer electron paramagnetic resonance. In unphosphorylated synthetic filaments, the effective rotational correlation times, tau(r), were 24 +/- 6 micros and 441 +/- 79 micros for the catalytic and regulatory domains, respectively. The corresponding amplitudes of motion were 42 +/- 4 degrees and 24 +/- 9 degrees as determined from steady-state phosphorescence anisotropy. These results suggest that the two domains have independent mobility due to a hinge between the two domains. Although a similar hinge was observed for skeletal myosin (Adhikari and Fajer (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 9643-9647. Brown et al. (2001) Biochemistry 40, 8283-8291), the latter displayed higher regulatory domain mobility, tau(r)= 40 +/- 3 micros, suggesting a smooth muscle specific mechanism of constraining regulatory domain dynamics. In the myosin monomers the correlation times for both domains were the same (approximately 4 micros) for both smooth and skeletal myosin, suggesting that the motional difference between the two isoforms in the filaments was not due to intrinsic variation of hinge stiffness. Heavy chain/regulatory light chain chimeras of smooth and skeletal myosin pinpointed the origin of the restriction to the heavy chain and established correlation between the regulatory domain dynamics with the ability of myosin to switch off but not to switch on the ATPase and the actin sliding velocity. Phosphorylation of smooth muscle myosin filaments caused a small increase in the amplitude of motion of the regulatory domain (from 24 +/- 4 degrees to 36 +/- 7 degrees ) but did not significantly affect the rotational correlation time of the regulatory domain (441 to 408 micros) or the catalytic domain (24 to 17 micros). These data are not consistent with a stable interaction between the two catalytic domains in unphosphorylated smooth muscle myosin filaments in the absence of nucleotides.

Effective rotational correlation times of proteins from NMR relaxation interference

Knowledge of the effective rotational correlation times, τ c , for the modulation of anisotropic spin–spin interactions in macromolecules subject to Brownian motion in solution is of key interest for the practice of NMR spectroscopy in structural biology. The value of τ c enables an estimate of the NMR spin relaxation rates, and indicates possible aggregation of the macromolecular species. This paper reports a novel NMR pulse scheme, [ 15N, 1H]-TRACT, which is based on transverse relaxation-optimized spectroscopy and permits to determine τ c for 15N– 1H bonds without interference from dipole–dipole coupling of the amide proton with remote protons. [ 15N, 1H]-TRACT is highly efficient since only a series of one-dimensional NMR spectra need to be recorded. Its use is suggested for a quick estimate of the rotational correlation time, to monitor sample quality and to determine optimal parameters for complex multidimensional NMR experiments. Practical applications are illustrated with the 110 kDa 7,8-dihydroneopterin aldolase from Staphylococcus aureus, the uniformly 15N-labeled Escherichia coli outer membrane protein X (OmpX) in 60 kDa mixed OmpX/DHPC micelles with approximately 90 molecules of unlabeled 1,2-dihexanoyl- sn-glycero-3-phosphocholine (DHPC), and the 16 kDa pheromone-binding protein from Bombyx mori, which cover a wide range of correlation times.

Spin label EPR studies of the effect of gramicidin S on lipid chain dynamics

The effect of gramicidin S (GS) on acyl chain dynamics of 14-doxylstearic acid spin label (14-SASL) monitoring the centre of dimyristoylphosphatidylcholine (DMPC) bilayers was studied with electron paramagnetic resonance (EPR) spectroscopy. The chain-melting transition of DMPC was shifted down and broadened monotonously by several degrees with increasing peptide concentration in the range of lipid/peptide ratios of l/p=5/1–15/1. The effective rotational correlation time τ eff of 14-SASL became larger with increasing amounts of peptide bound to fluid membranes of DMPC. From the dependence of τ eff on the lipid/peptide molar ratio it was estimated that about 10 DMPC molecules per monomer GS are involved in non-covalent peptide–lipid interaction. Optimized spectral subtractions showed that, well above the chain-melting transition, 90% of the EPR spectral intensity of the spin-labeled peptide–lipid complex (l/p=5/1) originated from spectra recorded in bilayers (without peptide) at lower temperature. This was not the case 6°C above the phase transition where two components with comparable intensities were detected. The two components were identified as different motional modes of the label molecules, rather than distinct mobile/immobile lipid populations.

Temperature Dependence of the Deuterium Quadrupole Coupling Constants and the Correlation Times for Neat Formamide

High-level ab initio calculations yield a linear relationship between the proton chemical shift, δH, and the deuterium quadrupole coupling constant, χD, for the amide protons in neat, liquid formamide. An experimental measurement of δH in combination with the linear relationships provides an accurate value for χD in the hydrogen-bonded liquid; subsequent measurements of the deuterium relaxation rate provide a means to obtain effective rotational correlation times in the neat liquid. Temperature-dependent proton chemical shifts and relaxation rates for the three nuclei in formamide are presented. The chemical shift data and the ab initio calculations indicate that both amide protons hydrogen bond, whereas the C−H proton does not participate in hydrogen bonding in the neat liquid. The effective correlation times at room temperature were found to be 9.2 ps for the trans N−D vector, 6.3 ps for the cis N−D vector, and 5.2 ps for the C−D vector. This indicates that in the neat liquid formamide rotates anisotropically.

Functional and structural differences in skeletal and cardiac myosins. A molecular dynamic approach

Conventional and saturation transfer electron paramagnetic resonance spectroscopy and differential scanning calorimetry were used to study the internal dynamics and stability of cardiac myosin. Intact and LC 2-deficient myosin isolated from bovine heart were spin-labelled with maleimide and iodoacetamide probe molecules at the SH1 sites. It was found that the probe molecules rotate with an effective rotational correlation time of 42 ns, which is at least six times shorter than the rotational correlation time of the same label on skeletal myosin. Addition of MgADP induces intrinsic changes in the multisubunit structure of myosin, but it does not lead to changes of the overall rotational properties of the myosin head. Temperature dependence of the EPR spectra of maleimide-labelled myosin shows continuous decrease of the spectral parameters (intensity ratio of the peak heights, hyperfine splitting) at increasing temperature. However, marked changes were obtained at about 16°C in LC 2-deficient myosin. DSC measurements also support the view that the removal of the LC 2 light chain produces change in the internal structure of cardiac myosin.

NMR assignments and secondary structure of the UvrC binding domain of UvrB

The 55 residue C-terminal domain of UvrB that interacts with UvrC during excision repair in Escherichia coli has been expressed and purified as a (His) 6 fusion construct. The fragment forms a stable folded domain in solution. Heteronuclear NMR experiments were used to obtain extensive 15N, 13C and 1H NMR assignments. NOESY and chemical shift data showed that the protein comprises two helices from residues 630 to 648 and from 652 to 670. 15N relaxation data also show that the first 11 and last three residues are unstructured. The effective rotational correlation time within the structured region is not consistent with a monomer. This oligomerisation may be relevant to the mode of dimerisation of UvrB with the homologous domain of UvrC.

Microsecond rotational dynamics of spin-labeled myosin regulatory light chain induced by relaxation and contraction of scallop muscle.

We have used saturation transfer electron paramagnetic resonance (ST-EPR) to study the rotational dynamics of spin-labeled regulatory light chain (RLC) in scallop (Placopecten magellanicus) muscle fibers. The single cysteine (Cys 51) in isolated clam (Mercenaria) RLC was labeled with an indanedione spin label (InVSL). RLC was completely and specifically extracted from scallop striated muscle fibers, eliminating the Ca sensitivity of ATPase activity and isometric force, which were both completely restored by stoichiometric incorporation of labeled RLC. The EPR spectrum of the isolated RLC revealed nanosecond rotational motions within the RLC, which were completely eliminated when the labeled RLC was bound to myosin heads in myofibrils or fibers in rigor. This is the most strongly immobilized RLC-bound probe reported to date and thus offers the most reliable detection of the overall rotational motion of the LC domain. Conventional EPR spectra of oriented fibers indicated essentially complete probe disorder, independent of ATP and Ca, eliminating orientational dependence and thus making this probe ideal for unambiguous measurement of microsecond rotational motions of the LC domain by ST-EPR. ST-EPR spectra of fibers in rigor indicated an effective rotational correlation time (taureff) of 140 +/- 5 microseconds, similar to that observed for the same spin label bound to the catalytic domain. Relaxation by ATP induced microsecond rotational motion (taureff = 70 +/- 4 microseconds), and this motion was slightly slower upon Ca activation of isometric contraction (taureff = 100 +/- 5 microseconds). These motions in relaxation and contraction are similar to, but slower than, the motions previously reported for the same spin label bound to the catalytic domain. These results support a model for force generation involving rotational motion of the LC domain relative to the catalytic domain and dynamic disorder-to-order transitions in both domains.

Dynamical behavior of the HIV-1 nucleocapsid protein

The HIV-1 nucleocapsid protein (NC) contains two CCHC-type zinc knuckle domains that are essential for genome recognition, packaging and infectivity. The solution structure of the protein has been determined independently by three groups. Although the structures of the individual zinc knuckle domains are similar, two of the studies indicated that the knuckles behave as independently folded, non-interacting domains connected by a flexible tether, whereas one study revealed the presence of interknuckle NOE cross-peaks, which were interpreted in terms of a more compact structure in which the knuckles are in close proximity. We have collected multidimensional NMR data for the recombinant, isotopically labeled HIV-1 NC protein, and confirmed the presence of weak interknuckle NOEs. However, the NOE data are not consistent with a single protein conformation. 15N NMR relaxation studies reveal that the two zinc knuckle domains possess different effective rotational correlation times, indicating that the knuckles are not tumbling as a single globular domain. In addition, the 1H NMR chemical shifts of isolated zinc knuckle peptides are very similar to those of the intact protein. The combined results indicate that the interknuckle interactions, which involve the close approach of the side-chains of Phe16 and Trp37, are transitory. The solution behavior of NC may be best considered as a rapid equilibrium between conformations with weakly interacting and non-interacting knuckle domains. This inherent conformational flexibility may be functionally important, enabling adaptive binding of NC to different recognition elements within the HIV-1 Ψ-RNA packaging signal.

Independent mobility of catalytic and regulatory domains of myosin heads.

The recent determination of the myosin head atomic structure has led to a new model of muscle contraction, according to which mechanical torque is generated in the catalytic domain and amplified by the lever arm made of the regulatory domain [Fisher, A. J., Smith, C. A., Thoden, J., Smith, R., Sutoh, K., Holden, H. M. & Rayment, I. (1995) Biochemistry 34, 8960-8972]. A crucial aspect of this model is the ability of the regulatory domain to move independently of the catalytic domain. Saturation transfer-EPR measurements of mobility of these two domains in myosin filaments give strong support for this notion. The catalytic domain of the myosin head was labeled at Cys-707 with indane dione spin label; the regulatory domain was labeled at the single cysteine residue of the essential light chain and exchanged into myosin. The mobility of the regulatory domain in myosin filaments was characterized by an effective rotational correlation time (tauR) between 24 and 48 micros. In contrast, the mobility of the catalytic domain was found to be tauR = 5-9 micros. This difference in mobility between the two domains existed only in the filament form of myosin. In the monomeric form, or when bound to actin, the mobility of the two domains in myosin was indistinguishable, with tauR = 1-4 micros and >1,000 micros, respectively. Therefore, the observed difference in filaments cannot be ascribed to differences in local conformations of the spin-labeled sites. The most straightforward interpretation suggests a flexible hinge between the two domains, which would have to stiffen before force could be generated.

Rotational mobility of Ca 2+-ATPase of sarcoplasmic reticulum in viscous media

The rotational diffusion of Ca 2+-ATPase [Ca 2+,Mg 2+-activated ATP phosphohydrolase E.C. 3.6.1.38] was studied in native sarcoplasmic reticulum membrane by saturation transfer ESR spectroscopy after covalent labelling of intramembranous sulfhydryl groups with nitroxyl derivative of maleimide (5-MSL) as a function of sucrose and glycerol in the suspending medium. The relative enzymatic activity of sarcoplasmic reticulum was followed by increasing the viscosity of the aqueous phase. The ATP hydrolysing activity of the enzyme decreased differently on adding sucrose and glycerol. In the case of sucrose the reciprocal of power dependence of viscosity was observed, whereas for glycerol an exponential decay law was obtained, indicating solvent-protein interaction. On increasing the viscosity of the aqueous phase by either sucrose or glycerol, no changes were observed in the intramembranous viscosity as measured using intercalated spin-labelled stearic acid (16-SASL). The effective rotational correlation time of the protein was measured, as a mobility parameter, using saturation transfer ESR spectroscopy and found to be increased linearly with the viscosity of the sucrose containing medium and for the extramembranous size a height of 6.8 nm was obtained, indicating that approx. 82% of the volume of Ca 2+-ATPase protein is external to the sarcoplasmic reticulum. The addition of glycerol probably promoted protein–protein interaction, as indicated by the larger changes in rotational diffusion and non-linear viscosity dependence.

Global topology & stability and local structure & dynamics in a synthetic spin-labeled four-helix bundle protein.

A maleimide nitroxide spin-label (MAL-6) linked to a cysteine in the hydrophobic core and a coproporphyrin I (CP) appended on the N-terminus of a synthetic helix-loop-helix peptide ([alpha2]) have been used to examine the designed self-association of a four-helix bundle ([alpha2]2), focusing on the bundle topology and stability and the rotational dynamics of the spin-label. Gel-permeation chromatography demonstrated that the [alpha2] peptide and the peptide modified with a spin-label ([MAL-6-alpha2]), a coproporphyrin ([CP-alpha2]) and a coproporphyrin plus a spin-label ([CP-MAL-6-alpha2]) self-associate into four helix bundles in solution as designed. Circular dichroism (CD) spectra prove that all these peptides are highly alpha-helical, confirmed for [alpha2]2 by Fourier transform infrared (FTIR) spectroscopic analysis. Electron spin resonance (ESR) spectra of the two attached maleimide spin-labels in [MAL-6-alpha2]2 shows their effective rotational correlation time (tau(c)) is 7.3 +/- 0.5 ns, consistent with that expected for the tumbling of the four helix bundle itself, indicating the labels are immobilized. The ESR spectra were also unaltered by aqueous-phase paramagnetic ions, Ni(II), demonstrating all of the spin-labels are buried within the hydrophobic core. The lack of spin-spin interaction between the buried, immobilized spin-labels indicates they are remote (> 15 A) from each other, indicating an antiparallel topology of the monomers in [MAL-6-alpha2]2. The parent [alpha2]2 and the modified [MAL-6-alpha2]2 and [CP-alpha2]2 peptides are highly stable (deltaG(H2O) approximately 25 kcal/mol) as investigated by guanidine hydrochloride denaturation curves monitored by ESR and CD spectroscopies. Guanidine hydrochloride denaturation leads to a shorter correlation time of the spin-label, tau(c) < 1 ns, approaching that of an unrestricted spin-label in solution. In contrast, trifluoroethanol caused dissociation of [MAL-6-alpha2]2 to yield two [MAL-6-alpha2] monomers with retention of secondary structure and changed the tau(c) to 2.5 +/- 0.5 ns, indicating that a significant degree of motional restriction is imposed on the spin-label by the secondary structure. The coproporphyrin probes covalently attached to the N-termini of [CP-alpha2]2 and [CP-MAL-6-alpha2]2 provided evidence that the helical monomers of both were in a parallel orientation, in contrast to the antiparallel orientation determined for [MAL-6-alpha2]2. Consequently, the ESR spectra of [MAL-6-alpha2]2 and [CP-MAL-6-alpha2]2 reveal major structural differences in the local vicinity of the spin-labels due to the topological difference between these two bundles. The ESR spectra of [CP-MAL-6-alpha2]2 contains two distinct nitroxide populations, indicating that one spin-label remains buried in the hydrophobic core and the other is excluded to solvent in this parallel topology. Alleviation of the steric interactions causing one spin-label in [CP-MAL-6-alpha2]2 to be solvent-exposed by addition of [CP-alpha2]2 results in formation of the heterodimeric [CP-alpha2]/[CP-MAL-6-alpha2], as evidenced by insertion of all the spin-labels into hydrophobic cores. The changes in global topology and local structure as evidenced by this pair of spectral probes have relatively minor effects on the course of guanidine denaturation of these bundles.

Internal flexibility of cardiac myosins

Conventional and saturation transfer electron paramagnetic resonance spectroscopy (EPR and ST EPR) and differential scanning calorimetry (DSC) were used to study the motional dynamics and segmental flexibility of cardiac myosins. Cardiac myosins isolated from bovine and human heart muscle were spin-labelled with isothiocyanate- or maleimide-based probe molecules at the reactive sulfhydryl sites (Cys-697 and Cys-707) of the motor domain. The maleimide probe molecules attached to human cardiac myosin rotated with an effective rotational correlation time of 33 ns which was at least eight times shorter than the rotational correlation time of the same label on skeletal myosin (260 ns). In the presence of MgADP and MgADP plus orthovanadate, flexibility changes in the multisubunit structure of myosins were detected, but this did not lead to changes of the overall rotational property of the myosin heads. Significant difference in the internal flexibility was detected on myosin samples isolated from ischemic tissue, the rotational correlation time decreased to 25 ns. DSC measurements supported the view that addition of nucleotides produced additional loosening in the multisubunit structure of cardiac myosin. It is postulated that there is an intersite communication between the nucleotide binding domain and the 20 kDa subunit where the reactive thiol sites are located.

Syntheses and Transglutaminase-Catalyzed Incorporation of Novel Spin-Labeled Primary Amines into Proteins

A series of spin-labeled primary amine derivatives, namely, 2,2,6,6-tetramethyl-piperidinyl-1-oxyl-4-amidoalkylamines with varying alkyl chain lengths, have been synthesized. The spin-labeled primary amine with a tetramethylene or a pentamethylene chain covalently modifies human plasma fibronectin with a stoichiometry of 0.97-to-1.0 (probe-to-subunit) in the presence of coagulation factor XIIIa. The labels with two or one methylene chain also similarly modify fibronectin, but with a stoichiometry of only about 0.3–0.4 per subunit. The spin-labeled primary amine with a trimethylene chain does not label fibronectin. The labeling site appears to be the glutamine-3 residue at the amino-terminal region of fibronectin. Electron spin resonance studies show that the bound labels are partially immobilized with an effective rotational correlation time of 0.4–0.6 ns. The spin-labeled primary amine with tetramethylene chain also is shown to covalently incorporate into bee venom melittin in the presence of guinea-pig liver transglutaminase. The syntheses of the various spin-labeled primary amines and their applications in the study of structure and dynamics of different proteins and peptides are discussed. The observations from this study suggest that these spin-labeled primary amines have potentially wide application as structural probes.