Structural biology workflow for the expression and characterization of functional human sodium glucose transporter type 1 in Pichia pastoris.

Heterologous expression of human membrane proteins is a challenge in structural biology towards drug discovery. Here we report a complete expression and purification process of a functional human sodium/D-glucose co-transporter 1 (hSGLT1) in Pichia pastoris as representative example of a useful strategy for any human membrane protein. hSGLT1 gene was cloned in two different plasmids to develop parallel strategies: one which includes green fluorescent protein fusion for screening optimal conditions, and another for large scale protein production for structural biology and biophysics studies. Our strategy yields at least 1 mg of monodisperse purified recombinant hSGLT1 per liter of culture, which can be characterized by circular dichroism and infrared spectroscopy as an alpha-helical fold protein. This purified hSGLT1 transports co-substrates (Na+ and glucose) and it is inhibited by phlorizin in electrophysiological experiments performed in planar lipid membranes.

Forearm pronation efficiency in A.L. 288-1 (Australopithecus afarensis) and MH2 (Australopithecus sediba): Insights into their locomotor and manipulative habits.

OBJECTIVES: The locomotor and manipulative abilities of australopithecines are highly debated in the paleoanthropological context. Australopithecus afarensis and Australopithecus sediba likely engaged in arboreal locomotion and, especially the latter, in certain activities implying manipulation. Nevertheless, their degree of arboreality and the relevance of their manipulative skills remain unclear. Here we calculate the pronation efficiency of the forearm (Erot ) in these taxa to explore their arboreal and manipulative capabilities using a biomechanical approach. MATERIALS AND METHODS: Three-dimensional humeral images and upper limb measurements of A.L. 288-1 (Au. afarensis) and MH2 (Au. sediba) were used to calculate Erot using a previously described biomechanical model. RESULTS: Maximal Erot in elbow flexion occurs in a rather supinated position of the forearm in Au. afarensis, similarly to Pan troglodytes. In elbow extension, maximal Erot in this fossil taxon occurs in the same forearm position as in Pongo spp. In Au. sediba the forearm positions where Erot is maximal are largely coincident with those for Hylobatidae. CONCLUSIONS: The pattern in Au. afarensis suggests relevant arboreal capabilities, which would include vertical climbing, although it is suggestive of poorer manipulative skills than in modern humans. The similarity between Au. sediba and Hylobatidae is difficult to interpret, but the differences between Au. sediba and Au. afarensis suggest that the capacity to rotate the forearm followed different evolutionary processes in these australopithecine species. Although functional inferences from the upper limb are complex, the observed differences between both taxa point to the existence of two distinct anatomical models.

Activity-related sexual dimorphism in Alaskan foragers from Point Hope: Evidences from the upper limb.

Ipiutak (100BCE-500CE) and Tigara (1200 - 1700CE) are two populations from Point Hope, Alaska. As commonly observed in forager communities, it may be expected males and females to have been involved in markedly different daily activities. Nevertheless, activity-related sexual dimorphism in these populations has been scarcely studied. Using humeral diaphyseal cross-sectional properties and forearm rotational efficiency, which are activity-dependent characteristics, we aim to assess differences between sexes and discuss what activities could have triggered them. Our results suggest that in Ipiutak males and females did not differ meaningfully in their cross-sectional properties. Conversely, in Tigara males had a greater rigidity of the entire humeral diaphysis than females, which suggests the existence of greater relative activity levels and more physically demanding tasks, possibly related to hunting activities. Concerning the differences between sexes in the forearm rotational efficiency, in Tigara females rotational efficiency in elbow flexion is maximal in a more supinated position than in males, which leads to an improvement of efficiency in those stages related to manipulation, and so improves the manipulative capacities of the upper limb. These differences in efficiency are caused by a more proximally oriented humeral medial epicondyle in females, which is thus confirmed to be a good feature to assess differences in labor. Therefore females in Tigara probably performed in a daily basis household activities, such as hide processing and other manipulative labors. In Ipiutak, the analysis of forearm rotational efficiency did not reveal differences between sexes. Overall, the results suggest that division of labor in Ipiutak was not as marked as in Tigara, where upper limb skeletal structure supports the idea that both sexes were involved in different daily activities. Nevertheless, the generalized lack of results in Ipiutak could be due to the small sample size, and thus interpretations should be considered with caution.

Analysis of the forearm rotational efficiency in extant hominoids: new insights into the functional implications of upper limb skeletal structure.

The greatly diversified locomotor behaviors in the Hominoidea impose different mechanical requirements in the upper limb of each species. As forearm rotation has a major role in locomotion, the skeletal structures involved in this movement may display differences among taxa that reflect functional adaptations. To test this, we use a biomechanical model that quantifies the rotatory capacity of pronator teres (rotational efficiency) from skeletal measurements. Using a large sample of hominoids, we aim to identify the morphological adaptations that confer differences in the mechanics of forearm motion and to assess the functional advantage of these adaptations. Forearm positions along the pronation-supination range where rotational efficiency is maximal depend on the orientation of the humeral medial epicondyle and differ among taxa. Our results indicate that these are related to locomotor mode. Knuckle-walkers exhibit a medial epicondyle more posteriorly directed, which, in elbow angles close to extension, causes rotational efficiency to be maximal in pronated positions of the forearm. Species with a significant amount of arboreal locomotion, such as vertical climbing, i.e., Pongo spp., Pan troglodytes and Gorilla gorilla, display more proximally oriented epicondyles, which, in elbow flexion, leads to maximum rotational efficiencies in supinated positions of the forearm. Hylobatidae, with the less posteriorly and proximally oriented epicondyle, show their maximum rotational efficiencies closer to the forearm neutral position throughout most of the flexion-extension range, which may be linked to brachiation in this taxon. In humans, the epicondylar orientation and thus the positions of the maximum rotational efficiencies fall between arboreal and terrestrial hominoids. This may be related to the enhanced manipulative skills of the upper limb. In conclusion, the current analysis indicates that the orientation of the humeral medial epicondyle is linked to the locomotor habits of extant hominoids and therefore can be used for locomotor inferences in fossil taxa.

Biomechanics of forearm rotation: force and efficiency of pronator teres.

Biomechanical models are useful to assess the effect of muscular forces on bone structure. Using skeletal remains, we analyze pronator teres rotational efficiency and its force components throughout the entire flexion-extension and pronation-supination ranges by means of a new biomechanical model and 3D imaging techniques, and we explore the relationship between these parameters and skeletal structure. The results show that maximal efficiency is the highest in full elbow flexion and is close to forearm neutral position for each elbow angle. The vertical component of pronator teres force is the highest among all components and is greater in pronation and elbow extension. The radial component becomes negative in pronation and reaches lower values as the elbow flexes. Both components could enhance radial curvature, especially in pronation. The model also enables to calculate efficiency and force components simulating changes in osteometric parameters. An increase of radial curvature improves efficiency and displaces the position where the radial component becomes negative towards the end of pronation. A more proximal location of pronator teres radial enthesis and a larger humeral medial epicondyle increase efficiency and displace the position where this component becomes negative towards forearm neutral position, which enhances radial curvature. Efficiency is also affected by medial epicondylar orientation and carrying angle. Moreover, reaching an object and bringing it close to the face in a close-to-neutral position improve efficiency and entail an equilibrium between the forces affecting the elbow joint stability. When the upper-limb skeleton is used in positions of low efficiency, implying unbalanced force components, it undergoes plastic changes, which improve these parameters. These findings are useful for studies on ergonomics and orthopaedics, and the model could also be applied to fossil primates in order to infer their locomotor form. Moreover, activity patterns in human ancient populations could be deduced from parameters reported here.

Functional plasticity of the human humerus: shape, rigidity, and muscular entheses.

The relationship between the mechanical loading undergone by a bone and its form has been widely assumed as a premise in studies aiming to reconstruct behavioral patterns from skeletal remains. Nevertheless, this relationship is complex due to the existence of many factors affecting bone structure and form, and further research combining structural and shape characteristics is needed. Using two-block PLS, which is a test to analyze the covariance between two sets of variables, we aim to investigate the relationship between upper-limb entheseal changes, cross-sectional properties, and contour shape of the humeral diaphysis. Our results show that individuals with strongly marked entheseal changes have increased diaphyseal rigidities. Bending rigidities are mainly related to entheseal changes of muscles that cross the shoulder. Moreover, the entheseal changes of muscles that participate in the rotation of the arm are related to mediolaterally flatter and ventrodorsally broader humeral shapes in the mid-proximal diaphysis. In turn, this diaphyseal shape is related to diaphyseal rigidity, especially to bending loadings. The shape of the diaphysis of the rest of the humerus does not covary either with rigidity or with entheseal changes. The results indicate that large muscular scars, such as those found in the mid-proximal diaphyses, seem to be related to diaphyseal shape, whereas this relationship is not seen for areas with less direct influences of powerful muscles.

3D analysis of the forearm rotational efficiency variation in humans.

Pronosupination is a component of the hominoid orthograde corporal plane that enables primates to execute efficient and sure locomotion in their habitat and is an essential movement for the development of manipulative capacities. We analyze human variability in the rotational efficiency of the pronator teres muscle by applying the biomechanical model created by Galtés et al. (Am J Phys Anthropol 2008; 135:293-300; Am J Phys Anthropol 2009a; 140:589-594) to skeletal remains of a human sample (N = 29) and three nonhuman hominoid specimens (chimpanzee, gorilla, and orangutan) by means of 3D technology. We aim to examine whether there is a distinctive human pattern of rotational efficiency and determine which structural features of the upper-limb bones have the greatest influence on the determination of rotational efficiency. Our results show that the human pattern differs from efficiencies observed in nonhuman hominoids, which may be interpreted in the light of morphofunctional adaptations. We identify medial epicondylar form as the key structure of the upper-limb bones for the determination of the rotational efficiency of the forearm. Results indicate that the more medially projected epicondyle of nonhuman hominoids relative to humans leads to higher values of maximum rotational efficiency. Moreover, the orientation of the medial epicondyle determines the pronounced differences in the position of the maximum efficiencies in the pronosupination range between humans and the studied nonhuman hominoids. Proximodistal orientation of the medial epicondyle is suggested to be a more appropriate feature for distinguishing between humans and nonhuman hominoids than anteroposterior orientation and, therefore, for inferring behavioral aspects from skeletal remains and fossils of primate upper-limb bones.

Structural features of the C-terminus from the human neurokinin-1 receptor.

The neurokinin-1 receptor (NK1R) is a G-protein coupled receptor found in the central and peripheral nervous systems of vertebrates, and is responsible for many physiological processes. The C-terminus domain seems to be essential for coupling to the corresponding G-protein and ß-arrestin, and is important for receptor desensitization, internalization and recycling. We have focused our study on expression of the human NK1R (hNK1R) C-terminus in Escherichia coli, and its purification and characterization, in order to elucidate its structural properties. CD and Fourier transform infrared spectroscopy showed that the hNK1R C-terminus, rather than having a random structure, has well-defined secondary-structure patterns. The presence of three tyrosine residues in the primary sequence of the hNK1R C-terminus facilitated the use of UV and fluorescence spectroscopy techniques which revealed tyrosine fluorescence and UV absorption at anomalous wavelengths. In their entirety, the results show that the hNK1R C-terminus has clearly defined secondary (25% α-helix, 27% unordered structure and 48% ß-sheets and ß-turns) and tertiary structures which, it is believed, are tightly related to its multiple functions.

On-chip photoactivation of heterologously expressed rhodopsin allows kinetic analysis of G-protein signaling by surface plasmon resonance spectroscopy.

Surface plasmon resonance spectroscopy allows the study of protein interaction dynamics in real-time. Application of this technique to G-protein coupled receptors, the largest family of receptors involved in signal transduction, has been complicated by their low level of expression and the critical dependence of their native conformation on the hydrophobic transmembrane lipid environment. Here, we investigate and compare three different strategies to immobilize rhodopsin, a prototypical G-protein coupled receptor on a sensor chip surface using antibodies and a lectin for receptor capturing. By further probing of different experimental conditions (pH, detergent type) we identified the optimal factors to maintain rhodopsin in a functional conformation and extended this approach to recombinant rhodopsin that was heterologously expressed in COS cells. Functional operation of rhodopsin on the sensor chip surface was proven by its activation and subsequent light-stimulated G-protein coupling. The influence of these experimental parameters on the association and dissociation kinetics of G-protein receptor coupling was determined. Thereby, we found that the kinetics of G(t) interaction were not changed by the strategy of immobilization or the type of detergent. Regeneration of opsin directly on a chip allowed recycling of the immobilized native and recombinant receptor. Thus, the approach provides an experimental framework for choosing the most suitable conditions for the solubilization, immobilization, and for functional tests of rhodopsin on a biosensor surface.

Technical note: Forearm pronation efficiency analysis in skeletal remains.

This work presents an original methodology for analyzing forearm-pronation efficiency from skeletal remains and its variation with regard to changes in the elbow position. The methodology is based on a biomechanical model that defines rotational efficiency as a mathematical function expressing a geometrical relationship between the origin and insertion of the pronator teres. The methodology uses humeral distal epiphysis photography, from which the geometrical parameters for the efficiency calculus can be obtained. Rotational efficiency is analyzed in a human specimen and in a living nonhuman hominoid (Symphalangus syndactylus) for a full elbow extension (180 degrees) and an intermediate elbow position (90 degrees). In both specimens, the results show that this rotational-efficiency parameter varies throughout the entire rotational range and show a dependency on the elbow joint position. The rotational efficiency of the siamang's pronator teres is less affected by flexion of the forearm than that of the human. The fact that forearm-pronation efficiency can be inferred, even quantified, allows us to interpret more precisely the functional and evolutionary significance of upper-limb skeletal design in extant and fossil primate taxa.

Functional implications of radial diaphyseal curvature.

A recent study (Galtés et al.: Am J Phys Anthropol 135 (2008) 293-300) demonstrated that during pronation, pronator teres exerts a favorable force for radial lateral bending. On the basis of this finding, we hypothesized that the pattern of muscular loading exerted on the radius by this muscle might play a role as a mechanical stimulus involved in radial bowing. The current work relates the hypertrophy of the forearm muscles to the degree of lateral curvature of the radial diaphysis. The analysis is based on an original osteometrical index to estimate radial curvature, and it applies a visual reference method to grade the osteological appearance of 10 entheses of 104 radii from archaeological and contemporary samples. Using these morphological data as an indirect method to measure the association between muscular hypertrophy and bone curvature, this study reveals that the pattern of muscular loading exerted on the apex of the radial shaft by the pronator teres muscle may play an important role as a mechanical stimulus involved in diaphyseal bowing.

Biomechanical model of pronation efficiency: new insight into skeletal adaptation of the hominoid upper limb.

Despite considerable literature on the functional anatomy of the hominoid upper limb, there are no quantitative approaches relating to bone design and the resulting muscular-activity enhancement. The purpose of this study is to quantitatively analyze the relationship between the rotational efficiency of the pronator teres muscle and the design of the skeletal structures on which it acts. Using conventional scan images of a human forearm for three rotational positions, this study develops an original biomechanical model that defines rotational efficiency as a mathematical function expressing a geometrical relationship between the origin and insertion muscular sites. The results show that this parameter varies throughout the entire pronation range, being maximal when the forearm lies around its functional position. Moreover, the rotational-efficiency formula allows us to demonstrate, by several simulation conditions, that an improvement in pronation efficiency is derived from a large shaft radius curvature, a large humeral medial epicondyle, and a more proximal pronator teres radial attachment. The fact that forearm pronation efficiency can be inferred, even quantified, throughout the entire rotational range, by applying the biomechanical model developed here allows us to undertake anatomical approaches in the field of Evolutionary Anthropology, to interpret more precisely how skeletal design is related to upper-limb function in extant and fossil primate taxa.

Critical role of electrostatic interactions of amino acids at the cytoplasmic region of helices 3 and 6 in rhodopsin conformational properties and activation.

The cytoplasmic sides of transmembrane helices 3 and 6 of G-protein-coupled receptors are connected by a network of ionic interactions that play an important role in maintaining its inactive conformation. To investigate the role of such a network in rhodopsin structure and function, we have constructed single mutants at position 134 in helix 3 and at positions 247 and 251 in helix 6, as well as combinations of these to obtain double mutants involving the two helices. These mutants have been expressed in COS-1 cells, immunopurified using the rho-1D4 antibody, and studied by UV-visible spectrophotometry. Most of the single mutations did not affect chromophore formation, but double mutants, especially those involving the T251K mutant, resulted in low yield of protein and impaired 11-cis-retinal binding. Single mutants E134Q, E247Q, and E247A showed the ability to activate transducin in the dark, and E134Q and E247A enhanced activation upon illumination, with regard to wild-type rhodopsin. Mutations E247A and T251A (in E134Q/E247A and E134Q/T251A double mutants) resulted in enhanced activation compared with the single E134Q mutant in the dark. A role for Thr(251) in this network is proposed for the first time in rhodopsin. As a result of these mutations, alterations in the hydrogen bond interactions between the amino acid side chains at the cytoplasmic region of transmembrane helices 3 and 6 have been observed using molecular dynamics simulations. Our combined experimental and modeling results provide new insights into the details of the structural determinants of the conformational change ensuing photoactivation of rhodopsin.

Ca2+/recoverin dependent regulation of phosphorylation of the rhodopsin mutant R135L associated with retinitis pigmentosa.

No single molecular mechanism accounts for the effect of mutations in rhodopsin associated with retinitis pigmentosa. Here we report on the specific effect of a Ca2+/recoverin upon phosphorylation of the autosomal dominant retinitis pigmentosa R135L rhodopsin mutant. This mutant shows specific features like impaired G-protein signaling but enhanced phosphorylation in the shut-off process. We now report that R135L hyperphosphorylation by rhodopsin kinase is less efficiently inhibited by Ca2+/recoverin than wild-type rhodopsin. This suggests an involvement of Ca2+/recoverin into the molecular pathogenic effect of the mutation in retinitis pigmentosa which is the cause of rod photoreceptor cell degeneration. This new proposed role of Ca2+/recoverin may be one of the specific features of the proposed new Type III class or rhodopsin mutations associated with retinitis pigmentosa.

Effect of dodecyl maltoside detergent on rhodopsin stability and function.

Detergent-solubilized bovine rhodopsin produces mixed detergent/lipid/protein micelles. The effect of dodecyl maltoside detergent on the thermal stability of dark-state rhodopsin, and upon formation of the different intermediates after rhodopsin photobleaching (metarhodopsin II and metarhodopsin III), and upon transducin activation has been studied. No significant effect is observed for the thermal stability of dark-state rhodopsin in the range of detergent concentrations studied, but a decrease in the stability of metarhodopsin II and an increase in metarhodopsin III formation is observed with decreasing detergent concentrations. The transducin activation process is also affected by the presence of detergent indicating that this process is dependent on the lipid micro-environment and membrane fluidity, and this stresses the importance of the native lipid environment in rhodopsin normal function.

[Rhodopsin structure: some light into the shadows of retinal degenerations]. / Estructura de la rodopsina: luz en las sombras de las degeneraciones retinianas.

Retinitis pigmentosa is a group of retinal degenerative diseases, within the broad family of hereditary retinopathies, for which there is no cure at present. Mutations in different genes coding for proteins related to the metabolism of photoreceptor cells, and to the visual phototransduction cascade, are the cause of this disease. Rhodopsin, the photoreceptor protein responsible for light absorption--and key in the first stages of vision--is one of the most studied molecules of the retina. Mutations in the opsin gene account for about 25% of all cases of autosomal dominant retinitis pigmentosa. Recent crystallization of this receptor in its inactive dark state has revealed new structural details yielding further insights into the intra and intermolecular mechanismsin which the protein is involved as a result of its activation.Furthermore, the in vitro study of recombinant rhodopsins carrying mutations previously found in retinitis pigmentosa patients (by means of spectroscopic and functional techniques) has shed new light on the structural requirements for its correct function, as well as the molecular defects underlying the mechanism of photoreceptor cell death. In this study, the main findings of the recent investigations carried out in this field are presented. The relevant information obtained at the molecular level is bound to facilitate our understandingof the molecular processes that will allow suitable therapiesfor different retinal degenerative diseases, particularly retinitis pigmentosa, to be proposed.

Estructura de la rodopsina: luz en las sombras de las degeneraciones retinianas / Rhodopsin structure: some light into the shadows of retinal degenerations

La retinosis pigmentaria comprende un grupo muy importante de enfermedades degenerativas de la retina, dentro del amplio conjunto de las retinopatías hereditarias, para las cuales no existe curación en la actualidad. Las mutaciones en diversas proteínas relacionadas con el metabolismo de las células fotorreceptoras y el proceso de fototransducción visual se encuentran en el origen de esta enfermedad. La rodopsina, fotorreceptor responsable de la absorción de luz y proteína fundamental en las primeras etapas del proceso visual, es una de las moléculas más estudiadas de la retina. Las mutaciones en el gen de la opsina son responsables del 25 por ciento de todos los casos de la forma autosómica dominante de la retinosis pigmentaria. La reciente cristalización de este receptor en su estado inactivo ha puesto de manifiesto nuevos detalles estructurales que permiten avanzar en la comprensión de los mecanismos intra e intermoleculares en los que la proteína participa como consecuencia de su activación. Asimismo, el estudio in vitro -mediante diversas técnicas espectroscópicas y funcionales- de rodopsinas con mutaciones detectadas previamente en pacientes con retinosis pigmentaria permite entender cuáles deben ser los requerimientos estructurales mínimos de la molécula para su funcionamiento correcto, así como los defectos que subyacen en el mecanismo molecular desencadenante de la muerte de las células fotorreceptoras. En este trabajo se presentan los principales aspectos de las investigaciones realizadas en los últimos años en este campo. La información molecular obtenida ha de facilitar el avance en la obtención de posibles soluciones terapéuticas para diversas enfermedades degenerativas de la retina, en particular de la retinosis pigmentaria (AU)

Altered functionality in rhodopsin point mutants associated with retinitis pigmentosa.

Point mutations found in rhodopsin associated with the retinal degenerative disease retinitis pigmentosa have been expressed in mammalian COS-1 cells, purified, and characterised. The mutations characterised-most of them for the first time-have been Met44Thr, Gly114Asp, Arg135Leu, Val137Met, and Pro171Leu in the transmembrane domain; Leu328Pro and Ala346Pro in the C-terminal tail of the cytoplasmic domain; and Gly106Trp in the intradiscal domain. Several of these mutations cause misfolding which results in impaired 11-cis-retinal binding. Two of them, Met44Thr and Val137Met, show spectral and structural features similar to those of wild type rhodopsin (Type I mutants) but significantly increased transducin initial activation rates. We propose that, in the case of these mutants, abnormal functioning resulting in faster activation kinetics could also play a role in retinitis pigmentosa by altering the stoichiometric balance of the different proteins involved in the phototransduction biochemical reactions.

The eye photoreceptor protein rhodopsin. Structural implications for retinal disease.

Rhodopsin is the membrane receptor responsible for photoreception in the vertebrate retina. Its characteristic seven-transmembrane helical structural motif is today widely recognised as a paradigm in signal transduction. Rhodopsin and the phototransduction system are frequently used as structural and mechanistic models for the G-protein coupled receptor superfamily. Recent advances in the activation mechanism (as derived from the structural available data) and the implications for normal and pathological - in retinal disorders - visual function will be reviewed.