The Critical Role of N- and C-Terminal Contact in Protein Stability and Folding of a Family 10 Xylanase under Extreme Conditions

Stabilization strategies adopted by proteins under extreme conditions are very complex and involve various kinds of interactions. Recent studies have shown that a large proportion of proteins have their N- and C-terminal elements in close contact and suggested they play a role in protein folding and stability. However, the biological significance of this contact remains elusive.In the present study, we investigate the role of N- and C-terminal residue interaction using a family 10 xylanase (BSX) with a TIM-barrel structure that shows stability under high temperature, alkali pH, and protease and SDS treatment. Based on crystal structure, an aromatic cluster was identified that involves Phe4, Trp6 and Tyr343 holding the N- and C-terminus together; this is a unique and important feature of this protein that might be crucial for folding and stability under poly-extreme conditions. folding and activity. Alanine substitution with Phe4, Trp6 and Tyr343 drastically decreased stability under all parameters studied. Importantly, substitution of Phe4 with Trp increased stability in SDS treatment. Mass spectrometry results of limited proteolysis further demonstrated that the Arg344 residue is highly susceptible to trypsin digestion in sensitive mutants such as ΔF4, W6A and Y343A, suggesting again that disruption of the Phe4-Trp6-Tyr343 (F-W-Y) cluster destabilizes the N- and C-terminal interaction. Our results underscore the importance of N- and C-terminal contact through aromatic interactions in protein folding and stability under extreme conditions, and these results may be useful to improve the stability of other proteins under suboptimal conditions

Caracterização da curva do lactato sanguíneo e aplicabilidade do modelo Dmax durante protocolo progressivo em esteira rolante La caracterización de la curva del lactato sanguíneo y la pertinencia del Dmax durante el protocolo progresivo en la cinta rodante Characterization of the blood lactate curve and applicability of the Dmax model in a progressive protocol on treadmill

PROPÓSITO: Caracterizar o comportamento do lactato sanguíneo ([La]), durante protocolo progressivo em esteira rolante, e investigar a aplicabilidade do modelo Dmax na detecção do limiar de lactato (LL) e rendimento esportivo. MÉTODOS: Vinte e sete homens atletas de nível regional executaram protocolo de Heck et al. (1985), com incrementos a cada três minutos. O rendimento esportivo foi obtido pela velocidade média da prova de 10km. O 1º e 2º LL foram determinados através de análise visual da curva das [La] (LLv1 e LLv2) e por interpolação na velocidade referente às concentrações de 2,0 e 3,5mmol.l¹ (LL2,0 e LL3,5). O modelo Dmax identificou o LL em valores medidos (DmaxMED) e preditos pelas funções polinomial (DmaxPOL), linear de dois segmentos (DmaxSEG) e exponencial contínua (DmaxEXP). A característica do lactato sanguíneo durante o teste incremental foi verificada pelos ajustes linear de dois segmentos e exponencial contínua. RESULTADOS: Não houve diferença significativa entre o somatório dos resíduos quadrados dos ajustes de curva, porém, houve tendência de melhor ajuste exponencial contínua em 70,4% da amostra. Enquanto não houve diferença significativa entre os DmaxMED, DmaxPOL, DmaxSEG e DmaxEXP, os métodos Dmax foram maiores do que LLv1, menores do que LL3,5 e não diferentes de LL2,0. Todos os critérios Dmax foram significativamente menores do que a velocidade média da prova de 10km. CONCLUSÕES: Enquanto as [La] tenderam a um aumento exponencial durante protocolos progressivos em esteira rolante, o modelo Dmax apresentou evidências da sua aplicabilidade para a detecção do LL, mas não do rendimento esportivo.<br>PROPÓSITO: Este estudio tenía como los objetivos, para caracterizar la conducta del lactato sanguíneo ([La]), durante el protocolo progresivo en la cinta rodante, y para investigar la pertinencia del Dmax en el descubrimiento del umbral de lactato (LL) y el ingreso deportivo. MÉTODOS: Veintisiete atletas de nivel regional ejecutaron protocolo de Heck et al. (1985), con incrementos cada 3 minutos. El ingreso deportivo se obtuvo por la velocidad de la prueba de 10 km. El 1 y 2 LL sea cierto a través del análisis visual de la curva del [La] (LLv1 y LLv2), y para la interpolación en la velocidad con respecto a las concentraciones de 2,0 y 3,5 mmol.l-1 (LL2,0 y LL3,5). EL Dmax identificó LL en los valores moderados (DmaxMED), y se predijo por el polinomial de las funciones (DmaxPOL), lineal de dos segmentos (DmaxSEG), y exponencial continuo (DmaxEXP). La característica del lactato sanguíneo durante la prueba incremental se verificó por los ajustes lineal de 2 segmentos y exponencial continuo. RESULTADOS: No había diferencia significante entre el sumatoria de los residuos cuadrados de los ajustes de la curva, sin embargo, había una tendencia continua de ajuste exponencial bueno en 70,4% de la muestra. Mientras que no había diferencia significante entre DmaxMED, DmaxPOL, DmaxSEG y DmaxEXP, el método Dmax es más grande que LLv1, más pequeño que LL3,5, y no presenta diferencia con el de LL2,0. Todo el criterio Dmax sea significativamente más pequeño que la velocidad elemento de la prueba de 10 km. CONCLUSIONES: Mientras las [LA] tenderon a un aumento exponencial durante los protocolos progresivos en la cinta rodante, el Dmax ejemplar presentó evidencias de pertinencia mayor el descubrimiento de LL, pero no para rendimiento deportivo.<br>PURPOSE: To characterize the blood lactate ([La]) behavior along a progressive protocol on treadmill, and to investigate the applicability of the Dmax model in detecting the lactate threshold (LT) and the sportive performance. METHODS: Twenty-seven male athletes of regional level performed the Heck et al. protocol (1985) incremented every 3 minutes. The sportive output was attained by the mean velocity of the 10 km-test. The first and second LT were determined through visual analysis of the [La] (LTv1 and LTv2) curve, and by interpolation of the velocity related to the 2.0 and 3.5 mmol.l-1 concentrations (LT2.0 and LT3.5). The Dmax model has identified the LT in measured values (DmaxMED), and was predicted by the polynomial functions (DmaxPOL), the 2-segment linear (DmaxSEG) and the continuous exponential (DmaxEXP). The characteristic of the blood lactate along the incremental test was checked through 2-segment linear adjustments and continuous exponential. RESULTS: There was no significant difference between the sums of the square residues of the curve adjustments, but there was a trend for a better continuous exponential adjustment at 70.4% of the sampling. Although there was no significant difference between the DmaxMED, DmaxPOL, DmaxSEG, and DmaxEXP, the Dmax methods were higher than the LTv1, lower than the LT3.5, and were not different of the LT2.0. Every Dmax criteria were significantly lower than the mean velocity of the 10 km-test. CONCLUSIONS: While the [La] trended to an exponential increase along the progressive protocols on treadmill, the Dmax model presented evidences of its applicability to detect the LT, but not for the sportive output

Effect of acute normobaric hypoxia on the ventilatory threshold

PURPOSE: This study investigated the response of the ventilatory threshold (VT) to acute normobaric hypoxia and compared the agreement between software-based algorithms which use automatic detection to identify the VT. Results were used to examine whether the VT can be used as a physiological parameter to prescribe and monitor exercise intensity in hypoxic exercise training programs. METHODS: Fourteen untrained individuals (7 women, 7 men; age 22 ± 2 years, [Formula: see text]O2peak 46 ± 7 mL kg(-1) min(-1)) completed five identical graded exercise tests (randomized order) on a cycle ergometer to measure VT at sea-level (SL) and in response to four normobaric hypoxic conditions (FIO2: 0.185, 0.165, 0.142, 0.125) equivalent to 1,000, 2,000, 3,000 and 4,000 m. Data were analyzed using a one-way analysis of variance (ANOVA) with repeated measures. RESULTS: The VT was similar across all conditions (SL = 1.98 ± 0.46, 1,000 m = 2.03 ± 0.61, 2,000 m = 2.27 ± 0.62, 3,000 m = 1.84 ± 0.50, 4,000 m = 2.29 ± 0.58 L min(-1)) for all algorithms used despite a reduction in arterial oxygen saturation at 3,000 (P ≤ 0.01) and 4,000 m (P ≤ 0.01) compared with SL values. CONCLUSION: The VT appears to be a suitable physiological parameter for exercise prescription in normobaric hypoxia up to an altitude of 4,000 m