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PubMed1978

Anterior QRS loop in hypertrophic cardiomyopathy.

Frank vectorcardiograms (VCGs) were reviewed in 45 patients with hypertrophic cardiomyopathy (HCM), 26 with obstruction and 19 without obstruction. Twelve of the 19 patients without obstruction and five of the 26 patients with obstruction were found to have predominantly anterior QRS loops. Fourteen patients had a large left anterior QRS loop with increased anterior and leftward force; the posterior and terminal rightward force were within the normal range, and the T loops were displaced posteriorly and to the right opposite to the QRS loop. Asymmetric septal and apical hypertrophy were noted echocardiographically and/or angiographically. Increased electrical force from the asymmetric hypertrophy of the septal and apical area is proposed to explain this large left anterior loop. Three patients had a QRS loop located anteriorly and to the right with electrocardiograms (ECGs) resembling those of posterolateral myocardial infarction or right ventricular hypertrophy. These finding suggest that (1) hypertrophic cardiomyopathy may be another cause of an anterior QRS loop; (2) the recognition of the large left anterior loop in the VCG in patients with a left ventricular hypertrophy pattern in the ECG is helpful in the diagnosis of HCM, especially the nonobstructive form; and (3) hypertrophic cardiomyopathy should be considered in the differential diagnosis of myocardial infarction or right ventricular hypertrophy.

Adult↗
PubMed2001

Multiple intestinal 'loops' provide an in vivo model to analyse multiple mucosal immune responses.

Mucosal immunity plays an important role in preventing disease but the induction of protective mucosal immune responses remains a significant challenge. We describe a novel in vivo model to analyze the induction of multiple mucosal immune responses in the small intestine. A sterile segment of intestine ('intestinal-segment'; 2-3 m long) was surgically prepared in the jejunum of 4-6-month-old lambs. This 'intestinal-segment' was then subdivided into consecutive segments, designated as 'loops' (15-20 cm long), that included a Peyer's patch (PP), or 'interspaces' (15-70 cm long), that lacked a visible PP. All 'loops' were sterile when collected 1-4 weeks post-surgery and there was no macroscopic or histological evidence of altered lymph or blood flow. Flow cytometric analysis of cells isolated from PP, mucosal epithelium (IEL) and the lamina propria (LPL) revealed no significant alterations in the cell populations present in 'loop' tissues. The functional integrity of M-cell antigen uptake in sterile intestinal 'loops' was evaluated by comparing the immune response induced by varying doses of soluble versus particulate porcine serum albumin (PSA formulated in alginate microspheres). A dose-dependent, PSA-specific antibody-secreting cell response was restricted to PP present in 'loops' injected with particulate PSA. These observations suggested that PP present in sterile 'loops' were functional and this conclusion was confirmed by detecting cholera toxin-specific antibody-secreting cells and secreted antibody in PP and intestinal contents, respectively, of immunized 'loops.' Thus, each 'loop' provided an independent site to analyze antigen-uptake and the induction of mucosal immune responses by a variety of antigen or vaccine formulations.

Animal Population Groups↗
PubMedDate not supplied

A hypothesis to explain the substrate reactivity of ribosomal and stem-loop RNA with ricin A-chain.

A hypothesis is proposed which explains the low catalytic efficiency of ricin A-chain on artifical stem-loop RNA substrates, relative to the high catalytic efficiency found on intact mammalian ribosomes. The enzymatic binding energy required to reach the transition state is greater than that to stabilize the stem structure of stem-loop RNA molecules. When artifical stem-loop complexes bind, the base-pairing of the stem is lost rapidly relative to catalysis. Loss of secondary structure causes movement of the susceptible adenine to a catalytically unfavorable geometry and most of the enzyme-substrate complexes dissociate without catalysis. The protein architecture of intact ribosomes, when bound to ricin A-chain, is proposed to stabilize the stem-loop structure to maintain adenine 4324 in a configuration external to the RNA phosphodiester backbone. The slow catalysis observed with small stem-loop structures is a consequence of the relative probabilities for stem-melting induced by the enzyme and for reaching the transition state. Catalysis with a ten-base stem-loop RNA shows product formation at a rate of up to 0.02 hr-1, but fails to achieve a full catalytic turnover with long incubations. The substoichiometry of product formation with small stem-loops is proposed to be a consequence of the competing rates of catalysis and enzyme denaturation under in vitro assay conditions. Rates for these processes are estimated from the kinetic parameters for ricin A-chain hydrolysis of ribosomes and stem-loop structures.

Humans↗
PubMed1997

Uveal melanoma. Comparison of the prognostic value of fibrovascular loops, mean of the ten largest nucleoli, cell type, and tumor size.

PURPOSE: This study was performed to determine the prognostic significance of the presence of loops defined as periodic acid-Schiff-positive fibrovascular septa that completely surround lobules of tumor cells in cases of uveal melanoma. METHODS: The presence of loops was evaluated using an ordinary light microscope and routinely stained periodic acid-Schiff and hematoxylin sections from 496 posterior uveal melanomas without knowledge of the follow-up data on the patient. RESULTS: At 15 years, survival decreased from 67.5% to 33.8% when complete loops were present. Univariate Cox regression analysis indicated that the presence of loops was an indicator of poor outcome, and was better than age but not as good as the mean diameter of the largest nucleoli, cell type, or tumor size. CONCLUSIONS: The presence of loops, as evaluated in this study, was not as strong an indicator of poor outcome as were loops assessed in a previous study of 234 cases from another laboratory. The authors suspect this difference may be due to their only using routinely stained sections without a green filter, as was used in previously reported studies. The authors description of loops does not require any special equipment and gives sufficiently useful results to justify its inclusion by the pathologist in reports of such specimens. A description of vascular loops should be added to the use of the modified Callender cell type, tumor dimensions, mitotic count, extraocular extension, and lymphocytic infiltration in the final pathologic report.

Cell Division↗
PubMed1999

Postoperative intraocular lens rotation: a randomized comparison of plate and loop haptic implants.

OBJECTIVE: To compare the postoperative rotation of plate and loop haptic implants of spherical power to ascertain the optimal design appropriate for toric intraocular lenses (IOLs). DESIGN: Randomized, controlled trial. PARTICIPANTS: Forty-eight patients attending for routine cataract surgery by phacoemulsification. METHOD: Patients with cataract as the only ocular disease were randomly implanted with plate or loop haptic implants after uncomplicated phacoemulsification. The baseline position of the IOL was determined from a video frame acquired at the conclusion of surgery. Postoperative IOL position was documented using digital retroillumination images at 2 weeks and 6 months after surgery. Capsular fusion patterns were recorded using slit-lamp biomicroscopy. Correlation of IOL rotation with axial length, capsular contraction, and fusion was attempted. MAIN OUTCOME MEASURES: Early IOL rotation, occurring between surgery and 2 weeks after surgery, was graded as mild (<10 degrees), moderate (10 degrees < to <30 degrees), or severe (>30 degrees) by a semiobjective online comparison of the images. Late IOL rotation, occurring between 2 weeks and 6 months, was measured more precisely using software developed specifically for the study. RESULTS: Twenty-three patients were allocated the loop haptic and 25 the plate haptic IOL. The groups were comparable for demographic variables and numbers of patients excluded from analysis (P > 0.05). Five (24%) of 21 of plate haptic IOLs underwent severe early rotation compared to 2 (9%) of 22 loop haptics (P = 0.36). The median late rotation was 6.8 degrees for loop haptics compared to 0.6 degrees for plate haptics (P = 0.0073). Between 2 weeks and 6 months, anticlockwise rotation had occurred in 16 (89%) of 18 loop haptic IOLs compared to 11 (52%) of 21 plate haptic IOLs (P = 0.0081). CONCLUSIONS: Plate haptic IOLs show greater rotational stability than do loop haptics made from polypropylene once capsular fusion has taken place. Loop haptics invariably rotate anticlockwise after 2 weeks.

Capsulorhexis↗
PubMed2001

Significance of the internal locking mechanism for loop security enhancement in the arthroscopic knot.

PURPOSE: Secure tissue fixation of an arthroscopic knot depends on the security of the initial loop and additional locking half-hitches. The purpose of this report is to evaluate the loop and knot security relative to the internal locking mechanism of arthroscopic slip knot. TYPE OF STUDY: This is an experimental study designed for knot and loop security on a material testing system. The measurements were carried out in a blinded fashion in which the tester was not aware of the type of knot being tested. METHODS: Five configurations of arthroscopic knots (overhand throw, Duncan loop, Revo knot, Tennessee slider, and SMC knot) were created around a 5-mm diameter metal bar using a No. 2 braided suture. For each knot configuration, 10 knots were tested for displacement at cyclic loading, load to clinical failure (3-mm displacement), ultimate failure load, and mode of failure on the servo-hydraulic material testing system (MTS 858 MiniBionix test system; MTS, Minneapolis, MN). A 1-way analysis of variance was used to determine the statistical difference in displacement at cyclic loading, load to clinical failure, and ultimate failure load between each knot configuration. RESULTS: In the cyclic loading test, the average displacement of all knots was clinically insignificant, with the average being less than 0.24 mm. The overhand throw had the worst characteristic in the load to failure test. The other 4 knots had an optimal load to failure. The SMC knot, which has an internal locking mechanism, had good loop and knot security in the load to clinical failure and ultimate failure load (P <.05). The SMC and the Revo knots failed by knot breakage (material failure) whereas the other knots failed by knot slippage (loop failure). CONCLUSIONS: The internal locking mechanism flips the post strand to convert the loop strand into a new post strand while rerouting the original post strand around a new post. An arthroscopic knot with an internal locking mechanism can enhance loop security.

Analysis of Variance↗
PubMed1996

The effects of loops on the torsional stiffnesses of rectangular wires: an in vitro study.

Various loop designs have been investigated with respect to the increased torsional flexibility given to the wire by their inclusion. The torsional flexibility of the wire was shown to be enlarged from 26% to 63%, depending on loop design. Both variation in wire cross-section and loop geometry will influence torsional stiffness. Wires with small cross-sectional dimensions will show greater flexibility in torsion. Even small deviations from the stated cross-sectional dimensions can influence the change in torsional stiffness caused by a specific loop design. Inclusion of a reverse-closing loop led to the greatest increase in flexibility, while a Bull loop changed torsional stiffness the least. When vertical flexibility is needed along with reduced torsional stiffness, the T-loop is the loop of choice.

Analysis of Variance↗
PubMed2001

Investigation of the functional role of active site loop II in a hypoxanthine phosphoribosyltransferase.

Hypoxanthine phosphoribosyltransferases (HPRTs) are of biomedical interest because defects in the enzyme from humans can result in gouty arthritis or Lesch-Nyhan syndrome, and in parasites these enzymes are potential targets for antiparasite chemotherapy. In HPRTs, a long flexible loop (active site loop II) closes over the active site during the enzyme catalyzed reaction. Functional roles for this loop have been proposed but have yet to be substantiated. For the present study, seven amino acids were deleted from loop II of the HPRT from Trypanosoma cruzi to probe the functional role of this active site loop in catalysis. The mutant enzyme (Deltaloop II) was expressed in bacteria, purified by affinity chromatography, and kinetic constants were determined for substrates of both forward (purine salvage) and reverse (pyrophosphorolysis) reactions catalyzed by the enzyme. Loop II deletion resulted in moderate (0.6-2.7-fold) changes in the Michaelis constants (K(m)s) for substrates other than pyrophosphate (PP(i)), for which there was a 5.8-fold increase. In contrast, k(cat) values were severely affected by loop deletion, with rates that were 240-840-fold below those for the wild-type enzyme. Together with previously reported structural data, these results are consistent with active site loop II participating in transition-state stabilization by precise positioning of the substrates for in line nucleophilic attack and in the liberation of PP(i) as a product of the salvage reaction.

Animals↗
PubMed2003

P-loop catalytically assisting the enzymatic cleavage of single-stranded DNA.

We demonstrated that a P-loop, a looped complex formed inside duplex DNA by adding peptide nucleic acids (PNA), acts catalytically as a template for enzymatic cleavage of single-stranded probe oligodeoxynucleotides (ODN). A PD-loop complex formed from P-loop and probe ODN was digested efficiently by a restriction enzyme, and the truncated probe ODN was released. The P-loop nicked by the enzyme can form PD-loop again with another probe ODN, and then assisted the enzymatic cleavage of an excess of probe ODN. In addition, by using dumbbell-formed ODN as a probe ODN, the efficiency of the P-loop-assisted ODN cleavage was enhanced considerably as compared with that of linear ODN. Thus, the method utilizing P-loop will make it possible to amplify the sequence information of duplex DNA via a catalytic cleavage of probe ODNs.

Base Sequence↗
PubMed2000

Conformations of the rhodopsin third cytoplasmic loop grafted onto bacteriorhodopsin.

BACKGROUND: The third cytoplasmic loop of rhodopsin (Rho EF) is important in signal transduction from the retinal in rhodopsin to its G protein, transducin. This loop also interacts with rhodopsin kinase, which phosphorylates light-activated rhodopsin, and arrestin, which displaces transducin from light-activated phosphorylated rhodopsin. RESULTS: We replaced eight residues of the EF loop of bacteriorhodopsin (BR) with 24 residues from the third cytoplasmic loop of bovine Rho EF. The surfaces of purple membrane containing the mutant BR (called IIIN) were imaged by atomic force microscopy (AFM) under physiological conditions to a resolution of 0.5-0.7 nm. The crystallinity and extracellular surface of IIIN were not perturbed, and the cytoplasmic surface of IIIN increased in height compared with BR, consistent with the larger loop. Ten residues of Rho EF were excised by V8 protease, revealing helices E and F in the AFM topographs. Rho EF was modeled onto the BR structure, and the envelope derived from the AFM data of IIIN was used to select probable models. CONCLUSIONS: A likely conformation of Rho EF involves some extension of helices E and F, with the tip of the loop lying over helix C and projecting towards the C terminus. This is consistent with mutagenesis data showing the TTQ transducin-binding motif close to loop CD, and cysteine cross-linking data indicating the C-terminal part of Rho EF to be close to the CD loop.

Amino Acid Sequence↗
PubMed1999

Dual conformations for the HIV-1 gp120 V3 loop in complexes with different neutralizing fabs.

BACKGROUND: The third hypervariable (V3) loop of HIV-1 gp120 has been termed the principal neutralizing determinant (PND) of the virus and is involved in many aspects of virus infectivity. The V3 loop is required for viral entry into the cell via membrane fusion and is believed to interact with cell surface chemokine receptors on T cells and macrophages. Sequence changes in V3 can affect chemokine receptor usage, and can, therefore, modulate which types of cells are infected. Antibodies raised against peptides with V3 sequences can neutralize laboratory-adapted strains of the virus and inhibit syncytia formation. Fab fragments of these neutralizing antibodies in complex with V3 loop peptides have been studied by X-ray crystallography to determine the conformation of the V3 loop. RESULTS: We have determined three crystal structures of Fab 58.2, a broadly neutralizing antibody, in complex with one linear and two cyclic peptides the amino acid sequence of which comes from the MN isolate of the gp120 V3 loop. Although the peptide conformations are very similar for the linear and cyclic forms, they differ from that seen for the identical peptide bound to a different broadly neutralizing antibody, Fab 59.1, and for a similar peptide bound to the MN-specific Fab 50.1. The conformational difference in the peptide is localized around residues Gly-Pro-Gly-Arg, which are highly conserved in different HIV-1 isolates and are predicted to adopt a type II beta turn. CONCLUSIONS: The V3 loop can adopt at least two different conformations for the highly conserved Gly-Pro-Gly-Arg sequence at the tip of the loop. Thus, the HIV-1 V3 loop has some inherent conformational flexibility that may relate to its biological function.

Amino Acid Sequence↗
PubMed1997

An inverse correlation between loop length and stability in a four-helix-bundle protein.

BACKGROUND: The loops in proteins are less well characterized than the secondary structural elements that they connect. We have used the four-helix-bundle protein Rop as a model system in which to explore the role of loop length in protein folding and stability. RESULTS: A natural two-residue loop was replaced with a series of glycine linkers up to 10 residues in length. All 10 mutants are highly helical dimers that retain wild-type RNA-binding activity. As loop length is increased, the stability of Rop toward thermal and chemical denaturation is progressively decreased. CONCLUSIONS: All the mutants assume a wild-type-like structure, which suggests that the natural loop does not actively dictate the final protein fold. The strong inverse correlation observed between loop length and stability is well described by a simple polymer model in which the entropy of loop closure is the dominant energetic term. Our results emphasize the importance of optimization of loop length to successful protein design.

Cloning, Molecular↗
PubMed1993

Functionally important domains of the large hydrophilic loop of CP47 as probed by oligonucleotide-directed mutagenesis in Synechocystis sp. PCC 6803.

The chlorophyll a-binding protein CP47 serves as core antenna to photosystem II (PS II). The predicted topology of CP47 exhibits six membrane-spanning regions and a large hydrophilic loop (loop E) which roughly includes 200 residues (255-455) and is presumably exposed to the lumenal side of the thylakoid membrane. Several lines of experimental evidence suggest that loop E might be involved in binding or stabilizing functional manganese in the catalytic site of water oxidation or in interacting with the extrinsic PS II-O protein (the 33-kDa manganese-stabilizing protein). To scan loop E for functionally important domains, oligonucleotide-directed mutagenesis has been used to introduce deletions of 3-8 residues in conserved and charged regions of loop E. In addition, one single-site mutation of the only histidine present in loop E was created (H343L). Domains deleted in delta 1 (I265-F268), delta 2 (T271-K277), delta 4 (T304-L309), delta 5 (F311-N317), and delta 12 (D440-P447) are required for stable assembly of functional PS II complexes. Deletion of domains delta 3 (K277-E283) and delta 11 (R422-E428) significantly reduces the level of assembled PS II and impairs photoautotrophic growth and oxygen evolution. Deletion of domain delta 8 (A373-D380) enhances the susceptibility to photoinhibition. In contrast, deletion of domains delta 6 (G333-I336), delta 7 (K347-R352), delta 9 (V392-Q394), and delta 10 (D416-F420) and mutation of H343 to leucine do not seem to severly interrupt PS II structure and function, although all mutants exhibit a slightly decreased stability of PS II as compared to the wild type. Thus, selected domains of the large hydrophilic loop of CP47 are important for PS II structure and function. With respect to possible sites of interaction between loop E of CP47 and the extrinsic PS II-O protein, our results indicate that none of the deletions in the region from residue 330 to 420 (delta 6, delta 7, delta 8, delta 9, delta 10) completely interrupts a functional association of the manganese-stabilizing protein to PS II, although the binding characteristics might be changed in some cases.

Amino Acid Sequence↗
PubMed1992

Functional topology of a surface loop shielding the catalytic center in lipoprotein lipase.

Lipoprotein lipase (LPL), hepatic lipase, and pancreatic lipase show high sequence homology to one another. The crystal structure of pancreatic lipase suggests that it contains a trypsin-like Asp-His-Ser catalytic triad at the active center, which is shielded by a disulfide bridge-bounded surface loop that must be repositioned before the substrate can gain access to the catalytic residues. By sequence alignment, the homologous catalytic triad in LPL corresponds to Asp156-His241-Ser132, absolutely conserved residues, and the homologous surface loop to residues 217-238, a poorly conserved region. To verify these assignments, we expressed in vitro wild-type LPL and mutant LPLs having single amino acid mutations involving residue Asp156 (to His, Ser, Asn, Ala, Glu, or Gly), His241 (to Asn, Ala, Arg, Gln, or Trp), or Ser132 (to Gly, Ala, Thu, or Asp) individually. All 15 mutant LPLs were totally devoid of enzyme activity, while wild-type LPL and other mutant LPLs containing substitutions in other positions were fully active. We further replaced the 22-residue LPL loop which shields the catalytic center either partially (replacing 6 of 22 residues) or completely with the corresponding hepatic lipase loop. The partial loop-replacement chimeric LPL was found to be fully active, and the complete loop-replacement mutant had approximately 60% activity, although the primary sequence of the hepatic lipase loop is quite different. In contrast, replacement with the pancreatic lipase loop completely inactivated the enzyme. Our results are consistent with Asp156-His241-Ser132 being the catalytic triad in lipoprotein lipase.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Mutational and kinetic analysis of a mobile loop in tyrosyl-tRNA synthetase.

The role of a mobile loop in tyrosyl-tRNA synthetase has been investigated by mutating each amino acid in the loop and kinetically analyzing the effect that each mutation has on the formation of the enzyme-bound tyrosyl adenylate intermediate. Kinetic analyses of mutations at three of the nine positions in the loop, K230, K233, and T234, have been reported elsewhere (Fersht et al., 1988; First & Fersht, 1993a,b). In this paper, the kinetic analyses of mutants in the remaining six positions, as well as a mutant in which the entire loop is deleted, are reported. With the exception of E235, which stabilizes the E.-[Tyr-ATP]++ and E.Tyr.ATP complexes by 1.0 and 1.2 kcal/mol, respectively, none of the remaining amino acids appears to be directly involved in the catalytic mechanism of the enzyme. Instead, mutation of these residues results in small alterations in the stability of E.Tyr.ATP, E.[Tyr-ATP]++ and E.Tyr.AMP.PPi complexes. The precise amino acid residues which stabilize each state vary, suggesting that the loop adopts different conformations in each of the complexes with the most highly constrained conformation being in the E.[Tyr-ATP]++ complex. Deletion of the loop reveals that the net effect of the loop in catalysis is two-fold: (1) to destabilize the E.Tyr.ATP complex preceding formation of the E.[Tyr.ATP]++ complex and (2) to stabilize the E.[Tyr-ATP]++ complex, indicating that the involvement of the loop in catalysis occurs at the expense of ATP-binding energy.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1991

Conformational feasibility of a hairpin with two purines in the loop. 5'-d-GGTACIAGTACC-3'.

Structural feasibility and conformational requirements for the sequence 5'-d-GGTACIAGTACC-3' to adopt a hairpin loop with I6 and A7 in the loop are studied. It is shown that a hairpin loop containing only two nucleotides can readily be formed without any unusual torsional angles. Stacking is continued on the 5'-side of the loop, with the I6 stacked upon C5. The base A7, on the 3'-side of the loop, can either be partially stacked with I6 or stick outside without stacking. Loop closure can be achieved for both syn and anti conformations of the glycosidic torsions for G8 while maintaining the normal Watson-Crick base pairing with the opposite C5. All torsional angles in the stem fall within the standard B-family of DNA helical structures. The phosphodiesters of the loop have trans,trans conformations. Loop formation might require the torsion about the C4'-C5' bond of G8 to be trans as opposed to the gauche+ observed in B-DNA. These results are discussed in relation to melting temperature studies [Howard et al. (1991) Biochemistry (preceding paper in this issue)] that suggest the formation of very stable hairpin structures for this sequence.

Base Sequence↗
PubMed1985

Common structural framework of the two Ca2+/Mg2+ binding loops of troponin C and other Ca2+ binding proteins.

The refinement of the crystal structure of turkey skeletal muscle troponin C at 2.2-A resolution reveals that the two calcium binding loops that are occupied by Ca2+ ions adopt conformations very similar to those of the two homologous loops of parvalbumin and to that of loop III-IV of the intestinal calcium binding protein. This specific fold assures suitable spatial positioning of the Ca2+ ligands. It consists of two reverse turns, one located at each end of the loop, and four Asx turns (a cyclic hydrogen-bonded structure involving an oxygen of the side chain of residue n and the main-chain amide nitrogen of residue n + 2) whenever such a side chain coordinates to the metal ion. The fifth Ca2+ coordination position in both loops of troponin C is occupied by a water molecule that is within hydrogen-bonding distance of an aspartic acid, thus mediating indirect interaction between the cation and the negatively charged carboxylate. The same loop framework is conserved in the two Ca2+ binding loops of parvalbumin and loop III-IV of the intestinal Ca2+ binding protein in spite of the variability in the nature of the side chains at equivalent positions. The disposition of the Ca2+ and of its coordinating water molecule relative to the protein main chain is conserved in all these cases.

Amino Acid Sequence↗
PubMed2002

Light-induced changes in the structure and accessibility of the cytoplasmic loops of rhodopsin in the activated MII state.

Bovine rhodopsin was specifically labeled on the cytoplasmic surface at cysteine 140 (the first residue of the loop connecting helices III and IV) or at cysteine 316 (in the loop connecting helix VII and the palmitoylation sites) with the fluorescent labels fluorescein and Texas Red. These loops are involved in activation and signal transduction. The time-resolved fluorescence depolarization was measured in the dark state and in the M(II) state, with labeled samples consisting of rhodopsin-octylglucoside micelles or rod outer segment (ROS) membranes. In this way the diffusional dynamics of the flexible loops of rhodopsin were measured for the first time directly on the nanosecond time scale. Control experiments showed that the large number of weak excitation pulses required in these single photon counting experiments leads to <5% bleaching of the sample. Rhodopsin was trapped in the activated M(II) state for the duration of the fluorescence experiments ( approximately 20 min) after illumination at pH 6 and 5 degrees C. For both types of samples and at both labeled positions the dynamics of the label and loop motion as monitored by the time constants of the depolarization were not significantly different in the two states of the receptor. The end-anisotropy increased, however, from 0.09 in the dark to 0.16 in the M(II) state for ROS samples labeled at C140. The corresponding numbers for the C316 position are 0.06 and 0.12. Light-induced activation in M(II) is thus associated with a large increase in the loop steric hindrance due to a changed loop domain structure on the cytoplasmic surface. These results are supported by fluorescence quenching experiments with I(-), which indicate a significant decrease in the collisional quenching constant k(q) and in accessibility in the M(II) state at both positions. The rotational correlation time of the rhodopsin micelles increased from 48 ns in the dark state to 60 ns in M(II). This increase is caused by a change in volume and/or shape and is consistent with a structural change. These results demonstrate that time-resolved fluorescence depolarization is a powerful tool to study the changes in conformation and dynamics of the cytoplasmic loops that accompany the activation of rhodopsin and other G-protein coupled receptors.

Animals↗