Achieving consistency in the lateral nasal osteotomy during rhinoplasty: an external perforated technique.

The lateral nasal osteotomy is an integral element in rhinoplasty. A reproducible and predictable technique for the lateral nasal osteotomy (when indicated) is a significant contributor to operative success. A variety of methods and instrumentation are used to produce lateral osteotomies; currently, the two different modes used most frequently are the internal continuous and external perforated techniques. A previously published study by the senior author detailed the benefits of the external perforated osteotomy after comparing the two different methods. This article describes the role of the external perforated osteotomy technique in reproducing consistent results in rhinoplasty with minimal postoperative complications.

Clearing the smoke: the scientific rationale for tobacco abstention with plastic surgery.

The use of tobacco is a significant contributor to preventable morbidity and mortality in the United States. A significant proportion of cardiovascular diseases, various oral and pulmonary neoplasms, nonmalignant respiratory diseases, and peripheral vascular disorders can be attributed to the use of cigarettes. Surgical outcomes can also be adversely affected as a result of cigarette smoking with intraoperative and postoperative pulmonary, cardiovascular, and cerebrovascular complications as well as increased wound healing complications. These are found across the entire spectrum of surgical specialties. Tissue ischemia and wound-healing impairment secondary to the influence of tobacco is particularly problematic for the plastic surgeon, especially during elective facial aesthetic procedures, cosmetic and reconstructive breast operations, abdominoplasty, free-tissue transfer, and replantation procedures. By educating and providing guidelines to those patients who smoke and by refusing to operate on individuals who fail to abstain, tobacco-associated surgical morbidity in the plastic and reconstructive surgery patient can be eliminated.

Rationale for submucous resection of hypertrophied inferior turbinates in rhinoplasty: an evolution.

To achieve success in rhinoplasty, the plastic surgeon takes advantage of numerous intraoperative techniques designed to manipulate nasal soft tissue and the osseocartilaginous framework. Although the postoperative result may meet preoperative aesthetic goals, an element of nasal airway obstruction can persist from failure to acknowledge the role of inferior turbinates. Surgically responsive inferior turbinate hypertrophy is frequently not addressed secondary to inadequate history taking, incomplete physical examination, and/or surgeon reluctance to handle these sensitive structures. The goal of this article is to discuss the anatomy and physiology of the inferior turbinates, to present the role for inferior turbinate surgery during rhinoplasty, and to delineate the evolution of the current technique of submucosal resection of the inferior turbinates. Over the past 14 years, the senior author (R.J.R.) has performed inferior turbinates surgery on 648 patients as part of a rhinoplasty.

Activation of myosin light chain kinase requires translocation of bound calmodulin.

A novel translocation step is inferred from structural studies of the interactions between the intracellular calcium receptor protein calmodulin (CaM) and one of its regulatory targets. A mutant of CaM missing residues 2-8 (DeltaNCaM) binds skeletal muscle myosin light chain kinase with high affinity but fails to activate catalysis. Small angle x-ray scattering data reveal that DeltaNCaM occupies a position near the catalytic cleft in its complex with the kinase, whereas the native protein translocates to a position near the C-terminal end of the catalytic core. Thus, CaM residues 2-8 appear to facilitate movement of bound CaM away from the vicinity of the catalytic cleft.

Calmodulin remains extended upon binding to smooth muscle caldesmon: a combined small-angle scattering and fourier transform infrared spectroscopy study.

We show that calmodulin (CaM) has an extended conformation in its complexes with sequences from the smooth muscle thin filament protein caldesmon (CaD) by using small-angle X-ray and neutron scattering with contrast variation. The CaD sequences used in these experiments were a C-terminal fragment, 22kCaD, and a smaller peptide sequence within this fragment, MG56C. Each of these sequences contains the CaM-binding sites A and B previously shown to interact with the C- and N-terminal lobes of CaM, respectively [Wang et al. (1997) Biochemistry 36, 15026]. By modeling the scattering data, we show that the majority of the MG56C sequence binds to the N-terminal domain of CaM. FTIR data on CaM complexed with 22kCaD or with MG56C peptide show the 22kCaD sequence contains unordered, helix, and extended structures, and that the extended structures reside primarily in the MG56C portion of the sequence. There are small changes in secondary structure, involving approximately 12 residues, induced by CaM binding to CaD. These changes involve a net decrease in extended structures accompanied by an increase in alpha-helix, and they occur within the CaM and/or in the MG56C sequence.

The solution structure of the Sac7d/DNA complex: a small-angle X-ray scattering study.

Small-angle X-ray scattering has been used to study the structure of the multimeric complexes that form between double-stranded DNA and the archaeal chromatin protein Sac7d from Sulfolobus acidocaldarius. Scattering data from complexes of Sac7d with a defined 32-mer oligonucleotide, with poly[d(GC)], and with E. coli DNA indicate that the protein binds along the surface of an extended DNA structure. Molecular models of fully saturated Sac7d/DNA complexes were constructed using constraints from crystal structure and solution binding data. Conformational space was searched systematically by varying the parameters of the models within the constrained set to find the best fits between the X-ray scattering data and simulated scattering curves. The best fits were obtained for models composed of repeating segments of B-DNA with sharp kinks at contiguous protein binding sites. The results are consistent with extrapolation of the X-ray crystal structure of a 1:1 Sac7d/octanucleotide complex [Robinson, H., et al. (1998) Nature 392, 202-205] to polymeric DNA. The DNA conformation in our multimeric Sac7d/DNA model has the base pairs tilted by about 35 degrees and displaced 3 A from the helix axis. There is a large roll between two base pairs at the protein-induced kink site, resulting in an overall bending angle of about 70 degrees for Sac7d binding. Regularly repeating bends in the fully saturated complex result in a zigzag structure with negligible compaction of DNA. The Sac7d molecules in the model form a unique structure with two left-handed helical ribbons winding around the outside of the right-handed duplex DNA.

The assembly of immunoglobulin-like modules in titin: implications for muscle elasticity.

Titin, a giant muscle protein, forms filaments that span half of the sarcomere and cover, along their length, quite diversified functions. The region of titin located in the sarcomere I-band is believed to play a major rôle in extensibility and passive elasticity of muscle. In the I-band, the titin sequence contains tandem immunoglobulin-like (Ig) modules intercalated by a potentially non-globular region. By a combined approach making use of small angle X-ray scattering and nuclear magnetic resonance techniques, we have addressed the questions of what are the average mutual orientation of poly-Igs and the degree of flexibility around the domain interfaces. Various recombinant fragments containing one, two and four titin I-band tandem domains were analysed. The small-angle scattering data provide a picture of the domains in a mostly extended configuration with their long axes aligned head-to-tail. There is a small degree of bending and twisting of the modules with respect to each other that results in an overall shortening in their maximum linear dimension compared with that expected for the fully extended, linear configurations. This shortening is greatest for the four module construct ( approximately 15%). 15N NMR relaxation studies of one and two-domain constructs show that the motions around the interdomain connecting regions are restricted, suggesting that titin behaves as a row of beads connected by rigid hinges. The length of the residues in the interface seems to be the major determinant of the degree of flexibility. Possible implications of our results for the structure and function of titin in muscles are discussed.

Neutron-scattering studies reveal further details of the Ca2+/calmodulin-dependent activation mechanism of myosin light chain kinase.

Previously, we utilized small-angle X-ray scattering and neutron scattering with contrast variation to obtain the first low-resolution structure of 4Ca2+.calmodulin (CaM) complexed with a functional enzyme, an enzymatically active truncation mutant of skeletal muscle myosin light chain kinase (MLCK). These experiments showed that, upon binding to MLCK, CaM undergoes a conformational collapse identical to that observed when CaM binds to the isolated peptide corresponding to the CaM binding sequence of MLCK. CaM thereby was shown to release the inhibition of the kinase by inducing a significant movement of its CaM binding and autoinhibitory sequences away from the surface of the catalytic core [Krueger, J. K., Olah, G. A., Rokop, S. E., Zhi, G., Stull, J. T., and Trewhella, J. (1997) Biochemistry 36, 6017-6023]. We report here similar scattering experiments on the CaM.MLCK complex with the addition of substrates; a nonhydrolyzable analogue of adenosine-triphosphate, AMPPNP, and a peptide substrate for MLCK, a phosphorylation sequence from myosin regulatory light chain (pRLC). These substrates are shown to induce an overall compaction of the complex. The separation of the centers-of-mass of the CaM and MLCK components is shortened (by approximately 12 A), thus bringing CaM closer to the catalytic site compared to the complex without substrates. In addition, there appears to be a reorientation of CaM with respect to the kinase upon substrate binding that results in interactions between the N-terminal sequence of CaM and the kinase that were not observed in the complex without substrates. Finally, the kinase itself becomes more compact in the CaM.MLCK.pRLC.AMPPNP complex compared to the complex without substrates. This observed compaction of MLCK upon substrate binding is similar to that arising from the closure of the catalytic cleft in cAMP-dependent protein kinase upon binding pseudosubstrate.

Neutron and X-ray solution scattering provide insights into biomolecular structure and function.

Neutron and X-ray small-angle scattering techniques have made significant advances in their applications in structural molecular biology. They have become important tools for studying the structural basis for biomolecular function, revealing details of protein and DNA structure, as well as functionally important conformational flexibility and interactions. More powerful neutron and X-ray sources are now available which enable faster data acquisition on lower concentration samples, as well as time-resolved studies in the case of synchrotron sources. This source development has been accompanied by instrument development and advances in scattering techniques. At the same time, advances in molecular biology that facilitate preparation of samples have made available more biological molecules suitable for study by scattering techniques. In this review we briefly describe the basic theory and practice of small-angle scattering and follow with examples of its application to studying the conformations of biomolecules in solution, as well as within functional complexes.

Calmodulin binding to myosin light chain kinase begins at substoichiometric Ca2+ concentrations: a small-angle scattering study of binding and conformational transitions.

We have used small-angle scattering to study the calcium dependence of the interactions between calmodulin (CaM) and skeletal muscle myosin light chain kinase (MLCK), as well as the conformations of the complexes that form. Scattering data were measured from equimolar mixtures of a functional MLCK and CaM or a mutated CaM (B12QCaM) incompetent to bind Ca2+ in its N-terminal domain, with increasing Ca2+ concentrations. To evaluate differences between CaM-enzyme versus CaM-peptide interactions, similar Ca2+ titration experiments were performed using synthetic peptides based on the CaM-binding sequence from MLCK (MLCK-I). Our data show there are different determinants for CaM binding the isolated peptide sequence compared to CaM binding to the same sequences within the enzyme. For example, binding of either CaM or B12QCaM to the MLCK-I peptide is observed even in the presence of EGTA, whereas binding of CaM to the enzyme requires Ca2+. The peptide studies also show that the conformational collapse of CaM requires both the N and C domains of CaM to be competent for Ca2+ binding as well as interactions with each end of MLCK-I, and it occurs at approximately 2 mol of Ca2+/mol of CaM. We show that CaM binding to the MLCK enzyme begins at substoichiometric concentrations of Ca2+ (< or = 2 mol of Ca2+/mol of CaM), but that the final compact structure of CaM with the enzyme requires saturating Ca2+. In addition, MLCK enzyme does bind to 2Ca2+ x B12QCaM, although this complex is more extended than the complex with native CaM. Our results support the hypothesis that CaM regulation of MLCK involves an initial binding step at less than saturating Ca2+ concentrations and a subsequent activation step at higher Ca2+ concentrations.

Myosin light chain kinase: functional domains and structural motifs.

Conventional myosin light chain kinase found in differentiated smooth and non-muscle cells is a dedicated Ca2+/calmodulin-dependent protein kinase which phosphorylates the regulatory light chain of myosin II. This phosphorylation increases the actin-activated myosin ATPase activity and is thought to play major roles in a number of biological processes, including smooth muscle contraction. The catalytic domain contains residues on its surface that bind a regulatory segment resulting in autoinhibition through an intrasteric mechanism. When Ca2+/calmodulin binds, there is a marked displacement of the regulatory segment from the catalytic cleft allowing phosphorylation of myosin regulatory light chain. Kinase activity depends upon Ca2+/calmodulin binding not only to the canonical calmodulin-binding sequence but also to additional interactions between Ca2+/calmodulin and the catalytic core. Previous biochemical evidence shows myosin light chain kinase binds tightly to actomyosin containing filaments. The kinase has low-affinity myosin and actin binding sites in Ig-like motifs at the N- and C-terminus, respectively. Recent results show the N-terminus of myosin light chain kinase is responsible for filament binding in vivo. However, the apparent binding affinity is greater for smooth muscle myofilaments, purified thin filaments, or actin-containing filaments in permeable cells than for purified smooth muscle F-actin or actomyosin filaments from skeletal muscle. These results suggest a protein on actin thin filaments that may facilitate kinase binding. Myosin light chain kinase does not dissociate from filaments in the presence of Ca2+/calmodulin raising the interesting question as to how the kinase phosphorylates myosin in thick filaments if it is bound to actin-containing thin filaments.

Structures of calmodulin and a functional myosin light chain kinase in the activated complex: a neutron scattering study.

Calmodulin (CaM) is the major intracellular receptor for Ca2+ and is responsible for the Ca2+-dependent regulation of a wide variety of cellular processes via interactions with a diverse array of target enzymes. Our current view of the structural basis for CaM enzyme activation is based on biophysical studies of CaM complexed with small peptides that model CaM-binding domains. A major concern with interpreting data from these structures in terms of target enzyme activation mechanisms is that the larger enzyme structure might be expected to impose constraints on CaM binding. Full understanding of the molecular mechanism for CaM-dependent enzyme activation requires additional structural information on the interaction of CaM with functional enzymes. We have utilized small-angle X-ray scattering and neutron scattering with contrast variation to obtain the first structural view of CaM complexed with a functional enzyme, an enzymatically active truncation mutant of skeletal muscle myosin light chain kinase (MLCK). Our data show that CaM undergoes an unhindered conformational collapse upon binding MLCK and activates the enzyme by inducing a significant movement of the kinase's CaM binding and autoinhibitory sequences away from the surface of the catalytic core.

Intrasteric regulation of myosin light chain kinase.

Ca2+/calmodulin activates myosin light chain kinase by reversal of an autoinhibited state. The effects of substitution mutations on calmodulin activation properties implicate 4 of the 8 basic residues between the catalytic core and the calmodulin-binding domain in maintaining autoinhibition. These residues are further amino-terminal to the basic residues comprising the previously proposed pseudosubstrate sequence and suggest involvement of the connecting region in intrasteric autoinhibition. The pseudosubstrate model for autoinhibition proposes that basic residues within the autoinhibitory region mimic basic residues in the substrate and bind to defined acidic residues within the catalytic core. Charge reversal mutations of these specific acidic residues, however, had little or no effect on the Km value for regulatory light chain. From a total of 20 acidic residues on the surface of the substrate binding lobe of the catalytic core, 7 are implicated in binding directly or indirectly to the autoinhibitory domain but not to the light chain. Only 2 acidic residues near the catalytic site may bind to the autoinhibitory domain and the arginine at P-3 in the light chain. Exposure of these 2 residues upon calmodulin binding may be necessary and sufficient for light chain phosphorylation.

A 40-kilodalton protein binds specifically to an upstream sequence element essential for muscle-specific transcription of the human myoglobin promoter.

To define transcriptional control elements responsible for muscle-specific expression of the human myoglobin gene, we performed mutational analysis of upstream sequences (nucleotide positions -373 to +7 relative to the transcriptional start site) linked to a firefly luciferase gene. Transient expression assays in avian and mammalian cells indicated that a CCCACCCCC (CCAC box) sequence (-223 to -204) is necessary for muscle-specific transcription directed either by the native myoglobin promoter or by a heterologous minimal promoter linked to the myoglobin upstream enhancer region. A putative MEF2-like site (-160 to -169) was likewise necessary for full transcriptional activity in myotubes. Mutations within either of two CANNTG (E-box) motifs (-176 to -148) had only minimal effects on promoter function. We identified and partially purified from nuclear extracts a 40-kDa protein (CBF40) that binds specifically to oligonucleotides containing the CCAC box sequence. A mutation of the CCAC box that disrupted promoter function in vivo also impaired binding of CBF40 in vitro. These data suggest that cooperative interactions between CBF40 and other factors including MEF-2 are required for expression of the human myoglobin gene in skeletal muscle.

Evidence that the methylesterase of bacterial chemotaxis may be a serine hydrolase.

CheB, the methylesterase of chemotactic bacteria, catalyzes the hydrolysis of glutamyl-methyl esters in bacterial chemoreceptor proteins. The two cysteines predicted by the amino acid sequence of CheB were replaced by alanine residues. The resulting mutants, Cys207-Ala, Cys309-Ala and a double cysteine mutant Cys207-Ala/Cys309-Ala, retained methylesterase activity, indicating that sulfhydryls are not crucial for CheB mediated catalysis. A homology search revealed a conserved serine active-site region between residues 162 and 166 which is homologous to the active-site region of acetylcholine esterases, suggesting that Ser164 of CheB is the active-site nucleophile. Oligonucleotide-directed mutagenesis was used to change the serine to a cysteine. This Ser164-Cys mutant had less than 2% of the wild-type activity. Unlike the serine proteinases which utilize a 'catalytic triad' mechanism, CheB does not have the conserved histidine and aspartic acid residues located in positions N-terminal to the active-site serine. In addition, CheB is not labeled with di-isopropylfluorophosphate, a potent inhibitor of other serine hydrolases. A novel mechanism is proposed for CheB involving substrate-assisted catalysis to account for these apparent anomalies.