Volym och avsmalningsfunktioner för sitkagran (Picea sitchensis (Bong.) Carr.), gran (Picea abies (L.) Karst.), och hvitgran (Picea glauca (Moench) Voss) på Island

The aim of this study was to evaluate different types of volume and taper equations that can be used to predict single-tree stem volume and stem diameter at any given height along the tree stem for plantation grown Sitka spruce (Picea sitchensis (Bong.) Carr.), Norway spruce (Picea abies (L.) Karst) and White spruce (Picea glauca (Mounch) Voss) in Iceland. A number of published tree volume equations were tested and modified to predict the total stem volumes over bark but three logarithmic equations were taken for more in-depth analysis. Three taper equations were tested. Two variable-exponent equations (Kozak 1997, Kozak 2004) and one exponential equation described by Biging (1984). Data from a total of 617 sample trees were used in this study, collected from stands in various parts of the country and present different types of stands growing in different soil types and cover most of the site conditions suitable for forestry in Iceland. To fit the regression model for the volume equations an ordinary least-squares (OLS) method was used. Because the construction of taper equations requires longitudinal data or multiple measurements on individual trees, and violates the assumption of independence between observations, a mixed effects approach was used to model the within tree autocorrelation. Volume equation [5] which has breast height diameter (D), tree height (H) and (H-1.3) as independent variables gave the best results based on fit and validation statistics and are most suitable for all three species. In diameter prediction a modified version of the Biging (1984) equation gave the best results based on fit and validation statistics and is most suitable for all three species. In volume prediction the Biging (1984) equation showed some bias in predicting volume of small trees and the same was noticed for the equation developed by Kozak (1997). The equation developed by Kozak (2004) seems to be more flexible in predicting the volume of small trees as well as bigger trees and should give the best results in volume prediction among the taper equations.Syftet med denna studie har varit att utvärdera olika typer av volym och avsmalnings funktioner som kan användas för att förutsäga stamvolym och stamdiameter vid en viss höjd längs trädet för planterad Sitka gran (Picea sitchensis (Bong.) Carr.), gran (Picea abies (L.) Karst) och Vit gran (Picea glauca (Mounch) Voss) på Island. Ett antal publicerade volym samband testades och modifierades för att prediktera den totala stamvolymen på bark. De tre avsmalningsmodeller som utvärderades i studien var två variablaexponent modeller ( Kozak 1997, Kozak 2004) och en exponentiell modell beskriven av Biging (1984). Data från totalt 617 provträd ligger till grund för denna studie. Materialet har samlats in från bestånd i olika delar av landet och representerar olika ståndorter och omfattar merparten av markförhållanden lämplig för skogsbruk på Island. Skattningarna av parametrarna i volymsfunktionerna har utförts med regressionsanalys enligt minsta-kvadrat metoden(OLS). Eftersom avsmalningsfunktionerna baseras på data med flera mätningar på ett enskilt träd längs stammen kan vi inte utan vidare göra antagandet om oberoende mellan observationer i datat. För avsmalningsfunktionerna användes därför en blandad regressionsmodell med fixa parametrar och där trädindividen specificerades som en slumpmässig effekt. Volym ekvation [5], som har brösthöjdsdiameter (D), trädhöjd (H) och (H – 1.3) som oberoende variabler gav det bästa resultatet baserat på valideringsstatistiken och residualstudier för alla tre arterna. För avsmalningsfunktionerna gav Kozak 2004 det bästa resultatet för alla tre arterna. Skillnaderna mellan modellerna var dock små

The complex conformational dynamics of neuronal calcium sensor-1: A single molecule perspective

The human neuronal calcium sensor-1 (NCS-1) is a multispecific two-domain EF-hand protein expressed predominantly in neurons and is a member of the NCS protein family. Structure-function relationships of NCS-1 have been extensively studied showing that conformational dynamics linked to diverse ion-binding is important to its function. NCS-1 transduces Ca 2+ changes in neurons and is linked to a wide range of neuronal functions such as regulation of neurotransmitter release, voltage-gated Ca 2+ channels and neuronal outgrowth. Defective NCS-1 can be deleterious to cells and has been linked to serious neuronal disorders like autism. Here, we review recent studies describing at the single molecule level the structural and mechanistic details of the folding and misfolding processes of the non-myristoylated NCS-1. By manipulating one molecule at a time with optical tweezers, the conformational equilibria of the Ca 2+ -bound, Mg 2+ -bound and apo states of NCS-1 were investigated revealing a complex folding mechanism underlain by a rugged and multidimensional energy landscape. The molecular rearrangements that NCS-1 undergoes to transit from one conformation to another and the energetics of these reactions are tightly regulated by the binding of divalent ions (Ca 2+ and Mg 2+ ) to its EF-hands. At pathologically high Ca 2+ concentrations the protein sometimes follows non-productive misfolding pathways leading to kinetically trapped and potentially harmful misfolded conformations. We discuss the significance of these misfolding events as well as the role of inter-domain interactions in shaping the energy landscape and ultimately the biological function of NCS-1. The conformational equilibria of NCS-1 are also compared to those of calmodulin (CaM) and differences and similarities in the behavior of these proteins are rationalized in terms of structural properties

Direct single-molecule observation of calcium-dependent misfolding in human neuronal calcium sensor-1

Neurodegenerative disorders are strongly linked to protein misfolding, and crucial to their explication is a detailed understanding of the underlying structural rearrangements and pathways that govern the formation of misfolded states. Here we use single-molecule optical tweezers to monitor misfolding reactions of the human neuronal calcium sensor-1, a multispecific EF-hand protein involved in neurotransmitter release and linked to severe neurological diseases. We directly observed two misfolding trajectories leading to distinct kinetically trapped misfolded conformations. Both trajectories originate from an on-pathway intermediate state and compete with native folding in a calcium-dependent manner. The relative probability of the different trajectories could be affected by modulating the relaxation rate of applied force, demonstrating an unprecedented real-time control over the free-energy landscape of a protein. Constant-force experiments in combination with hidden Markov analysis revealed the free-energy landscape of the misfolding transitions under both physiological and pathological calcium concentrations. Remarkably for a calcium sensor, we found that higher calcium concentrations increased the lifetimes of the misfolded conformations, slowing productive folding to the native state. We propose a rugged, multidimensional energy landscape for neuronal calcium sensor-1 and speculate on a direct link between protein misfolding and calcium dysregulation that could play a role in neurodegeneration

Polyelectrolyte interactions enable rapid association and dissociation in high-affinity disordered protein complexes

Highly charged intrinsically disordered proteins can form complexes with very high affinity in which both binding partners fully retain their disorder and dynamics, exemplified by the positively charged linker histone H1.0 and its chaperone, the negatively charged prothymosin α. Their interaction exhibits another surprising feature: The association/dissociation kinetics switch from slow two-state-like exchange at low protein concentrations to fast exchange at higher, physiologically relevant concentrations. Here we show that this change in mechanism can be explained by the formation of transient ternary complexes favored at high protein concentrations that accelerate the exchange between bound and unbound populations by orders of magnitude. Molecular simulations show how the extreme disorder in such polyelectrolyte complexes facilitates (i) diffusion-limited binding, (ii) transient ternary complex formation, and (iii) fast exchange of monomers by competitive substitution, which together enable rapid kinetics. Biological polyelectrolytes thus have the potential to keep regulatory networks highly responsive even for interactions with extremely high affinities

Human Small Heat Shock Protein B8 Inhibits Protein Aggregation without Affecting the Native Folding Process

: Small Heat Shock Proteins (sHSPs) are key components of our Protein Quality Control system and are thought to act as reservoirs that neutralize irreversible protein aggregation. Yet, sHSPs can also act as sequestrases, promoting protein sequestration into aggregates, thus challenging our understanding of their exact mechanisms of action. Here, we employ optical tweezers to explore the mechanisms of action of the human small heat shock protein HSPB8 and its pathogenic mutant K141E, which is associated with neuromuscular disease. Through single-molecule manipulation experiments, we studied how HSPB8 and its K141E mutant affect the refolding and aggregation processes of the maltose binding protein. Our data show that HSPB8 selectively suppresses protein aggregation without affecting the native folding process. This anti-aggregation mechanism is distinct from previous models that rely on the stabilization of unfolded polypeptide chains or partially folded structures, as has been reported for other chaperones. Rather, it appears that HSPB8 selectively recognizes and binds to aggregated species formed at the early stages of aggregation, preventing them from growing into larger aggregated structures. Consistently, the K141E mutation specifically targets the affinity for aggregated structures without impacting native folding, and hence impairs its anti-aggregation activity

The Complex Conformational Dynamics of Neuronal Calcium Sensor-1: A Single Molecule Perspective

The human neuronal calcium sensor-1 (NCS-1) is a multispecific two-domain EF-hand protein expressed predominantly in neurons and is a member of the NCS protein family. Structure-function relationships of NCS-1 have been extensively studied showing that conformational dynamics linked to diverse ion-binding is important to its function. NCS-1 transduces Ca2+ changes in neurons and is linked to a wide range of neuronal functions such as regulation of neurotransmitter release, voltage-gated Ca2+ channels and neuronal outgrowth. Defective NCS-1 can be deleterious to cells and has been linked to serious neuronal disorders like autism. Here, we review recent studies describing at the single molecule level the structural and mechanistic details of the folding and misfolding processes of the non-myristoylated NCS-1. By manipulating one molecule at a time with optical tweezers, the conformational equilibria of the Ca2+-bound, Mg2+-bound and apo states of NCS-1 were investigated revealing a complex folding mechanism underlain by a rugged and multidimensional energy landscape. The molecular rearrangements that NCS-1 undergoes to transit from one conformation to another and the energetics of these reactions are tightly regulated by the binding of divalent ions (Ca2+ and Mg2+) to its EF-hands. At pathologically high Ca2+ concentrations the protein sometimes follows non-productive misfolding pathways leading to kinetically trapped and potentially harmful misfolded conformations. We discuss the significance of these misfolding events as well as the role of inter-domain interactions in shaping the energy landscape and ultimately the biological function of NCS-1. The conformational equilibria of NCS-1 are also compared to those of calmodulin (CaM) and differences and similarities in the behavior of these proteins are rationalized in terms of structural properties

Disorder in a two-domain neuronal Ca2+-binding protein regulates domain stability and dynamics using ligand mimicry.

Funder: Lundbeckfonden; doi: http://dx.doi.org/10.13039/501100003554Funder: Villum Fonden (DK)Understanding the interplay between sequence, structure and function of proteins has been complicated in recent years by the discovery of intrinsically disordered proteins (IDPs), which perform biological functions in the absence of a well-defined three-dimensional fold. Disordered protein sequences account for roughly 30% of the human proteome and in many proteins, disordered and ordered domains coexist. However, few studies have assessed how either feature affects the properties of the other. In this study, we examine the role of a disordered tail in the overall properties of the two-domain, calcium-sensing protein neuronal calcium sensor 1 (NCS-1). We show that loss of just six of the 190 residues at the flexible C-terminus is sufficient to severely affect stability, dynamics, and folding behavior of both ordered domains. We identify specific hydrophobic contacts mediated by the disordered tail that may be responsible for stabilizing the distal N-terminal domain. Moreover, sequence analyses indicate the presence of an LSL-motif in the tail that acts as a mimic of native ligands critical to the observed order-disorder communication. Removing the disordered tail leads to a shorter life-time of the ligand-bound complex likely originating from the observed destabilization. This close relationship between order and disorder may have important implications for how investigations into mixed systems are designed and opens up a novel avenue of drug targeting exploiting this type of behavior

Extreme disorder in an ultrahigh-affinity protein complex

Molecular communication in biology is mediated by protein interactions. According to the current paradigm, the specificity and affinity required for these interactions are encoded in the precise complementarity of binding interfaces. Even proteins that are disordered under physiological conditions or that contain large unstructured regions commonly interact with well-structured binding sites on other biomolecules. Here we demonstrate the existence of an unexpected interaction mechanism: the two intrinsically disordered human proteins histone H1 and its nuclear chaperone prothymosin-α associate in a complex with picomolar affinity, but fully retain their structural disorder, long-range flexibility and highly dynamic character. On the basis of closely integrated experiments and molecular simulations, we show that the interaction can be explained by the large opposite net charge of the two proteins, without requiring defined binding sites or interactions between specific individual residues. Proteome-wide sequence analysis suggests that this interaction mechanism may be abundant in eukaryotes