The role of cavities in protein dynamics: crystal structure of a photolytic intermediate of a mutant myoglobin.
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We determined the structure of the photolytic intermediate of a sperm whale myoglobin (Mb) mutant called Mb-YQR [Leu-(B10)-->Tyr; His(E7)-->Gln; Thr(E10)-->Arg] to 1.4-A resolution by ultra-low temperature (20 K) x-ray diffraction. Starting with the CO complex, illumination leads to photolysis of the Fe-CO bond, and migration of the photolyzed carbon monoxide (CO*) to a niche in the protein 8.1 A from the heme iron; this cavity corresponds to that hosting an atom of Xe when the crystal is equilibrated with xenon gas at 7 atmospheres [Tilton, R. F., Jr., Kuntz, I. D. & Petsko, G. A. (1984) Biochemistry 23, 2849-2857]. The site occupied by CO* corresponds to that predicted by molecular dynamics simulations previously carried out to account for the NO geminate rebinding of Mb-YQR observed in laser photolysis experiments at room temperature. This secondary docking site differs from the primary docking site identified by previous crystallographic studies on the photolyzed intermediate of wild-type sperm whale Mb performed at cryogenic temperatures [Teng et al. (1994) Nat. Struct. Biol. 1, 701-705] and room temperature [Srajer et al. (1996) Science 274, 1726-1729]. Our experiment shows that the pathway of a small molecule in its trajectory through a protein may be modified by site-directed mutagenesis, and that migration within the protein matrix to the active site involves a limited number of pre-existing cavities identified in the interior space of the protein. (+info)
Protein dynamics in an intermediate state of myoglobin: optical absorption, resonance Raman spectroscopy, and x-ray structure analysis.
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A metastable state of myoglobin is produced by reduction of metmyoglobin at low temperatures. This is done either by irradiation with x-rays at 80 K or by electron transfer from photoexcited tris(2, 2'-bipyridine)-ruthenium(II) at 20 K. At temperatures above 150 K, the conformational transition toward the equilibrium deoxymyoglobin is observed. X-ray crystallography, Raman spectroscopy, and temperature-dependent optical absorption spectroscopy show that the metastable state has a six-ligated iron low-spin center. The x-ray structure at 115K proves the similarity of the metastable state with metmyoglobin. The Raman spectra yield the high-frequency vibronic modes and give additional information about the distortion of the heme. Analysis of the temperature dependence of the line shape of the Soret band reveals that a relaxation within the metastable state starts at approximately 120 K. Parameters representative of static properties of the intermediate state are close to those of CO-ligated myoglobin, while parameters representative of dynamics are close to deoxymyoglobin. Thus within the metastable state the relaxation to the equilibrium is initiated by changes in the dynamic properties of the active site. (+info)
Heme orientation affects holo-myoglobin folding and unfolding kinetics.
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Native myoglobin (Mb) consists of two populations which differ in the orientation of the heme by 180 degrees rotation (as verified by nuclear magnetic resonance) but have identical absorption spectra and equilibrium-thermodynamic stability. Here, we report that these two fractions of native oxidized Mb (from horse) both unfold and refold (chemical denaturant, pH 7, 20 degrees C) in two parallel kinetic reactions with rate constants differing 10-fold. In accord, the oxidized heme remains coordinated to unfolded horse Mb in up to 4 M guanidine hydrochloride (pH 7, 20 degrees C). (+info)
Amide proton hydrogen exchange rates for sperm whale myoglobin obtained from 15N-1H NMR spectra.
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The hydrogen exchange behavior of exchangeable protons in proteins can provide important information for understanding the principles of protein structure and function. The positions and exchange rates of the slowly-exchanging amide protons in sperm whale myoglobin have been mapped using 15N-1H NMR spectroscopy. The slowest-exchanging amide protons are those that are hydrogen bonded in the longest helices, including members of the B, E, and H helices. Significant protection factors were observed also in the A, C, and G helices, and for a few residues in the D and F helices. Knowledge of the identity of slowly-exchanging amide protons forms the basis for the extensive quench-flow kinetic folding experiments that have been performed for myoglobin, and gives insights into the tertiary interactions and dynamics in the protein. (+info)
Sink or swim: strategies for cost-efficient diving by marine mammals.
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Locomotor activity by diving marine mammals is accomplished while breath-holding and often exceeds predicted aerobic capacities. Video sequences of freely diving seals and whales wearing submersible cameras reveal a behavioral strategy that improves energetic efficiency in these animals. Prolonged gliding (greater than 78% descent duration) occurred during dives exceeding 80 meters in depth. Gliding was attributed to buoyancy changes with lung compression at depth. By modifying locomotor patterns to take advantage of these physical changes, Weddell seals realized a 9.2 to 59.6% reduction in diving energetic costs. This energy-conserving strategy allows marine mammals to increase aerobic dive duration and achieve remarkable depths despite limited oxygen availability when submerged. (+info)
A fingerprinting method for chondroitin/dermatan sulfate and hyaluronan oligosaccharides.
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A previously published method for the analysis of glycosaminoglycan disaccharides by high pH anion exchange chromatography (Midura,R.J., Salustri,A., Calabro,A., Yanagishita,M. and Hascall,V.C. (1994), Glycobiology,4, 333-342) has been modified and calibrated for chondroitin and dermatan sulfate oligosaccharides up to hexasaccharide in size and hyaluronan oligosaccharides up to hexadecasaccharide. For hyaluronan oligosaccharides chain length controls elution position; however, for chondroitin and dermatan sulfate oligosaccharides elution times primarily depend upon the level of sulfation, although chain length and hence charge density plays a role. The sulfation position of GalNAc residues within an oligosaccharide is also important in determining its elution position. Compared to 4-sulfation a reducing terminal 6-sulfate retards elution; however, when present on an internal GalNAc residue it is the 4-sulfate containing oligosaccharide which elutes later. These effects allow discrimination between oligosaccharides differing only in the position of GalNAc sulfation. Using this simple methodology, a Dionex CarboPac PA-1 column with NaOH/NaCl eluents and detection by absorbance at 232 nm, a quantitative analytical fingerprint of a chondroitin/dermatan sulfate chain may be obtained, allowing a determination of the abundance of chondroitin sulfate, dermatan sulfate, and hyaluronan along with an analysis of structural features with a linear response to approximately 0.1 nmol. The method may readily be calibrated using either commercial disaccharides or the di- and tetrasaccharide products of a limit digest of commercial chondroitin sulfate by chondroitin ABC endolyase. Commercially available and freshly prepared shark, whale, bovine, and human cartilage chondroitin sulfates have been examined by this methodology and we have confirmed that freshly isolated shark cartilage CS contains significant amounts of the biologically important GlcA2Sbeta(1-3)GalNAc6S structure. (+info)
Observations on the muscles of the eye of the bowhead whale, Balaena mysticetus.
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The muscles of the eyelids and the extraocular muscles of mysticete whales are poorly described for a variety of reasons, including considerable difficulty in obtaining specimens. Our objective is to provide such a description for the bowhead whale, Balaena mysticetus. This study has examined the gross anatomy of the region in six specimens (five adults, one fetus) of the bowhead whale. Results show that the muscles associated with the eye are well developed and possess several distinctive features. For example, precise limits of each extraocular muscle are difficult to determine along their entire length because these muscles intermingle with one another near their insertion. Furthermore, some fibers from these muscles (except the retractor bulbi) also insert into the eyelids. Pulling on these muscles to simulate contraction results in movement of the eyelids and suggests a role for these muscles in palpebral retraction. Insertion of a large levator palpebrae superioris muscle into the upper eyelid further enhances opening of the palpebral fissure. Another unusual feature is the presence of tunnel-like structures that redirect the dorsal and ventral oblique muscles. The dorsal oblique muscle is redirected caudally about 90 degrees, then directed medially by another 90 degrees. These directional changes are accomplished via a connective tissue tunnel derived in part from the fibrous connective tissue of the dorsal rectus and the levator palpebrae superioris muscles. In most terrestrial mammals, a similar change in direction is accomplished by a cartilaginous trochlea. The ventral oblique muscle originates via a slender tendon from the frontal bone and undergoes a similar radical change in direction. Its tendon of insertion undergoes about a 90-degree change in direction that is accomplished through a tunnel-like structure derived from fibrous connective tissue of the ventral rectus muscle. Based on the morphology of the musculature presented, it is likely that the eyeballs and eyelids of the bowhead whale are quite mobile and appear capable of complex movement. The possibility of retraction and protrusion of the eyeball is discussed. (+info)
A novel two-over-two alpha-helical sandwich fold is characteristic of the truncated hemoglobin family.
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Small hemoproteins displaying amino acid sequences 20-40 residues shorter than (non-)vertebrate hemoglobins (Hbs) have recently been identified in several pathogenic and non-pathogenic unicellular organisms, and named 'truncated hemoglobins' (trHbs). They have been proposed to be involved not only in oxygen transport but also in other biological functions, such as protection against reactive nitrogen species, photosynthesis or to act as terminal oxidases. Crystal structures of trHbs from the ciliated protozoan Paramecium caudatum and the green unicellular alga Chlamydomonas eugametos show that the tertiary structure of both proteins is based on a 'two-over-two' alpha-helical sandwich, reflecting an unprecedented editing of the classical 'three-over-three' alpha-helical globin fold. Based on specific Gly-Gly motifs the tertiary structure accommodates the deletion of the N-terminal A-helix and replacement of the crucial heme-binding F-helix with an extended polypeptide loop. Additionally, concerted structural modifications allow burying of the heme group and define the distal site, which hosts a TyrB10, GlnE7 residue pair. A set of structural and amino acid sequence consensus rules for stabilizing the fold and the bound heme in the trHbs homology subfamily is deduced. (+info)